Cell Lysis...• Modern Proteomics –Sample Preparation,Analysis and Practical Applications...
Transcript of Cell Lysis...• Modern Proteomics –Sample Preparation,Analysis and Practical Applications...
Cell LysisA presentation of AFA-energeticsreg enabled Applications for high resolution sample preparation
Covaris
Table of Contents
Introduction 3
AFABenefits 3
High-throughputClinicalProteomicsfromCellsFreshFrozenTissueandFFPESamples 5
PreservingProteinIntegrityExtractionofNativeProteins 6
LowInputExtraction 8
Hard-to-lyseSamples 9
CellLysisinEukaryotes 9
CellLysisofPatientDerivedXenografts(PDXs) 10
CellLysisinProkaryotes 10
VersatilityofAFA 11
3
IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential
partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological
interpretation
Anidealsample-preparationmethodshould
bull Beasnon-selectiveaspossible
bull Preventlossandordegradationduringthepreparationprocedure
bull Avoidcontamination
bull Enablehigh-throughputprocessing
bull Ensurereproducibility
bull Becompatiblewiththedownstreamanalyticalmethod
TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample
Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques
likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming
CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe
workflowforMS
AFA Benefits
Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile
maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian
tissuesFFPEblocks)
Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe
targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe
veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich
requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples
ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom
difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas
nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading
tohigherqualityextractionandbiomarkerpreservation
AnalysisSampleCollection Cell Lysis ProcessingExtraction
bull Standardizesamplepreparation
bull Preservesampleheterogeneity
bull High-throughputautomationready
bull Reproducible
bull Compatiblewithdownstreamanalysismethods
bull Versatilemostsampletypes
bull Streamlineworkflow
4
References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist
etalIntJMolSci2015163537-3563DOI103390ijms16023537
bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages
84-98DOI101016jcca201604033
bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)
8ndash20DOI101016jjprot201608006
bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-
pp23-62ndash2017DOI101007978-3-319-41448-5_4
bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient
SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024
5
High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome
profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor
bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics
ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever
proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein
solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation
includingintegrationlaboratoryroboticsoftheprocess
AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics
TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111
ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the
abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto
extractandidentifyproteinsreproduciblyandconsistently
InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor
incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack
ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand
onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE
sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows
Keywords FFPE oncology cancer research paraffin
References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020
DOI101021acsjproteome9b00686JProtRes2020192973-983
- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)
overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years
bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal
httpsabrf2019gorgesappsusnode3876
- ThisworkisthefoundationoftheHYPER-solpaper
bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation
JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf
- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient
thisprotocolistoisolateproteinsthatcanbemissedbyothermethods
6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
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Table of Contents
Introduction 3
AFABenefits 3
High-throughputClinicalProteomicsfromCellsFreshFrozenTissueandFFPESamples 5
PreservingProteinIntegrityExtractionofNativeProteins 6
LowInputExtraction 8
Hard-to-lyseSamples 9
CellLysisinEukaryotes 9
CellLysisofPatientDerivedXenografts(PDXs) 10
CellLysisinProkaryotes 10
VersatilityofAFA 11
3
IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential
partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological
interpretation
Anidealsample-preparationmethodshould
bull Beasnon-selectiveaspossible
bull Preventlossandordegradationduringthepreparationprocedure
bull Avoidcontamination
bull Enablehigh-throughputprocessing
bull Ensurereproducibility
bull Becompatiblewiththedownstreamanalyticalmethod
TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample
Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques
likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming
CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe
workflowforMS
AFA Benefits
Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile
maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian
tissuesFFPEblocks)
Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe
targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe
veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich
requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples
ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom
difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas
nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading
tohigherqualityextractionandbiomarkerpreservation
AnalysisSampleCollection Cell Lysis ProcessingExtraction
bull Standardizesamplepreparation
bull Preservesampleheterogeneity
bull High-throughputautomationready
bull Reproducible
bull Compatiblewithdownstreamanalysismethods
bull Versatilemostsampletypes
bull Streamlineworkflow
4
References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist
etalIntJMolSci2015163537-3563DOI103390ijms16023537
bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages
84-98DOI101016jcca201604033
bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)
8ndash20DOI101016jjprot201608006
bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-
pp23-62ndash2017DOI101007978-3-319-41448-5_4
bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient
SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024
5
High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome
profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor
bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics
ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever
proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein
solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation
includingintegrationlaboratoryroboticsoftheprocess
AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics
TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111
ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the
abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto
extractandidentifyproteinsreproduciblyandconsistently
InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor
incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack
ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand
onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE
sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows
Keywords FFPE oncology cancer research paraffin
References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020
DOI101021acsjproteome9b00686JProtRes2020192973-983
- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)
overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years
bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal
httpsabrf2019gorgesappsusnode3876
- ThisworkisthefoundationoftheHYPER-solpaper
bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation
JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf
- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient
thisprotocolistoisolateproteinsthatcanbemissedbyothermethods
6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
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3
IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential
partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological
interpretation
Anidealsample-preparationmethodshould
bull Beasnon-selectiveaspossible
bull Preventlossandordegradationduringthepreparationprocedure
bull Avoidcontamination
bull Enablehigh-throughputprocessing
bull Ensurereproducibility
bull Becompatiblewiththedownstreamanalyticalmethod
TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample
Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques
likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming
CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe
workflowforMS
AFA Benefits
Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile
maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian
tissuesFFPEblocks)
Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe
targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe
veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich
requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples
ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom
difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas
nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading
tohigherqualityextractionandbiomarkerpreservation
AnalysisSampleCollection Cell Lysis ProcessingExtraction
bull Standardizesamplepreparation
bull Preservesampleheterogeneity
bull High-throughputautomationready
bull Reproducible
bull Compatiblewithdownstreamanalysismethods
bull Versatilemostsampletypes
bull Streamlineworkflow
4
References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist
etalIntJMolSci2015163537-3563DOI103390ijms16023537
bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages
84-98DOI101016jcca201604033
bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)
8ndash20DOI101016jjprot201608006
bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-
pp23-62ndash2017DOI101007978-3-319-41448-5_4
bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient
SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024
5
High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome
profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor
bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics
ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever
proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein
solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation
includingintegrationlaboratoryroboticsoftheprocess
AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics
TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111
ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the
abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto
extractandidentifyproteinsreproduciblyandconsistently
InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor
incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack
ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand
onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE
sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows
Keywords FFPE oncology cancer research paraffin
References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020
DOI101021acsjproteome9b00686JProtRes2020192973-983
- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)
overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years
bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal
httpsabrf2019gorgesappsusnode3876
- ThisworkisthefoundationoftheHYPER-solpaper
bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation
JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf
- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient
thisprotocolistoisolateproteinsthatcanbemissedbyothermethods
6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
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4
References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist
etalIntJMolSci2015163537-3563DOI103390ijms16023537
bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages
84-98DOI101016jcca201604033
bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)
8ndash20DOI101016jjprot201608006
bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-
pp23-62ndash2017DOI101007978-3-319-41448-5_4
bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient
SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024
5
High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome
profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor
bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics
ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever
proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein
solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation
includingintegrationlaboratoryroboticsoftheprocess
AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics
TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111
ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the
abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto
extractandidentifyproteinsreproduciblyandconsistently
InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor
incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack
ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand
onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE
sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows
Keywords FFPE oncology cancer research paraffin
References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020
DOI101021acsjproteome9b00686JProtRes2020192973-983
- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)
overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years
bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal
httpsabrf2019gorgesappsusnode3876
- ThisworkisthefoundationoftheHYPER-solpaper
bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation
JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf
- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient
thisprotocolistoisolateproteinsthatcanbemissedbyothermethods
6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
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5
High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome
profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor
bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics
ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever
proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein
solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation
includingintegrationlaboratoryroboticsoftheprocess
AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics
TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111
ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the
abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto
extractandidentifyproteinsreproduciblyandconsistently
InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor
incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack
ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand
onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE
sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows
Keywords FFPE oncology cancer research paraffin
References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020
DOI101021acsjproteome9b00686JProtRes2020192973-983
- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)
overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years
bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal
httpsabrf2019gorgesappsusnode3876
- ThisworkisthefoundationoftheHYPER-solpaper
bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation
JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf
- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient
thisprotocolistoisolateproteinsthatcanbemissedbyothermethods
6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
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6
Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind
conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications
communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational
modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas
examples
Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation
References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal
etalHUPO2012-poster
- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity
bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun
MassSpectrom2013272767ndash2776DOI101002rcm6742
- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike
phosphorylationandglycosylation
bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch
IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j
ijms201708015
- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues
bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)
3403ndash3408DOI101021acsjproteome5b00547
- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan
optimizeddetergentsystempermittedefficientnativeproteomeextraction
bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem
2016December155159ndash13DOI101016jab201609016
- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination
bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand
growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6
- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique
bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-
relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189
- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients
integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused
bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892
- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry
(XL-MS)
bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover
andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24
DOI101007s10585-013-9605-6
- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby
RPPAtechnique
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
- Button 20
- Button 21
- Button 22
- Button 23
7
bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof
GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006
- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis
bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion
monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017
Nov16(11)2006-2016DOI101074mcpRA117000023
- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof
thepre-requisites
bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative
proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384
- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey
hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome
enrichmentstepsuseddownstream
bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure
andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003
- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin
Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol
proteome
bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom
knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339
- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein
Chlamydomonasusingaquantitativelabel-freeapproach
bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-
NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472
- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand
resistancemechanismsinbreastcancer
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
- Button 20
- Button 21
- Button 22
- Button 23
8
Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel
canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable
toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina
sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube
willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers
havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso
calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization
Keywords low cell extraction low input cell lysis single cell
References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood
SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724
- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins
fromtheequivalentof100to200cells
bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal
Genome Biology201819161DOI101186s13059-018-1547-5
- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample
cleanupthatmayincursignificantlosses
bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof
enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c
- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)
bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-
2221DOI101083jcb201612051
- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to
understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells
bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass
SpectrometrySLietalposterndashASMS2013
- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction
techniques
9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
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9
Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker
recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand
theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin
processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle
ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative
proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85
DOIdxdoiorg101016jjprot201412016
- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites
bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal
ClinicalChemistry57111545-15552011DOI101373clinchem2010157743
- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP
fortissuepulverizationandAFAmethodforsuccessfulproteinextraction
bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-
ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288
DOI101016jdiagmicrobio201606001
- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria
bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002
pmic200800088
- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues
bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol
Prog200824606minus614DOI101021bp070359s
- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall
bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-
2840DOI101002jcb25933
- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue
bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal
BiotechnolProg2018Jan34(1)130-140DOI101002btpr255
- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused
Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery
efficientlyinawiderangeofapplications
bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome
Research200873035ndash3041DOI101021pr800012c
- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand
minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch
bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-
4684DOI101021pr400604b
- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother
conventionalmethodsrdquo
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
- Button 20
- Button 21
- Button 22
- Button 23
10
Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor
thispurpose
bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal
2017Aug810(491)DOI101126scisignalaam8065
- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast
xenografts
bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular
Proteomics15101074DOI101074mcpM114047480
- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof
proteinsandproteoforms
Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018
Jan196(1)DOI103390medsci6010008
- UseofAFAtodisruptS pneumoniaecapsule
bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits
BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109
No8August2012DOI101002bit24484
- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication
bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody
fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60
DOI101002bit24891
- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose
inthecontextofultrascaleddownoptimization
bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol
Prog271488ndash14962011DOI101002btpr644
- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
- Button 20
- Button 21
- Button 22
- Button 23
11
Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof
intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids
AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor
proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand
qualityoftrypticdigestionandforhydrogelssolubilization
Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)
References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918
62545-2558DOI101021acsjproteome9b00085
- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding
properproteinextractionandpreservationasexemplifiedbyAFA
bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-
AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692
DOI101021acsjproteome8b00295
- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as
allowedbymodernmassspectrometry
bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|
NUMBER7|JULY2017DOI101038ng3866
- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA
forefficientandreproduciblecelllysisproteinextractionandchromatinshearing
bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp
TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510
- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin
bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom
wpwp-contentuploads202007ASMS_2020_Posterpdf
- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe
processdownto20minutes
bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF
THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338
- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication
bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry
HanetalJPeriodontRes20181ndash10DOI101111jre12604
- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations
bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom
StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis
KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f
- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite
profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans
bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof
TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004
- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst
12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
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12
preparationsteps
bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260
(December2017)DOI102144000114617
- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs
bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash
1021DOI101016jcell201801032
- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments
bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110
DOI101038s41467-017-02618-6
- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments
bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell
PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141
- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment
bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer
Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61
- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked
toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using
cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels
Information subject to change without notice For research only Not for use in diagnostic procedures
USATel+17819323959|Emailcustomerservicecovariscom|EuropeTel+44(0)8458720100|Emailemeacustomerservicecovariscom|APAC+8613764276714|EmailAPACcustomerservicecomWebwwwcovariscom|Applicationsapplicationsupportcovariscom|ServiceandSupporttechsupportcovariscom|M020103_RevE_Apr2020|2020copyCovarisInc
Stay Connected
- Button 19
- Button 20
- Button 21
- Button 22
- Button 23