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Proteomics service laboratory

Just launched

Operated by

Karel Harant

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Recent develompent in proteomics• Rapid development of instrumentation• Decline of MALDI-based instrumenst• 2D protein electrophoresis abandoned• Development of many quantification techniques

• Emploing of data-independent acquisition and quantification

• Label-free quantification

• More than half of human proteome from one sample injection

• 80% of proteomic data acquired in last two years (Somebody, HUPO2014)

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Bottom-up proteomics

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Data processing• Tens of thousands MS2 spectra from 1hour gradient• Computational processing• Most comon approach

• Generation of theoretical mass list• Compare real precursor MS1 mass with theoretical precursor

mass• If there is match - compare theoretical and real MS2 spectra• No sequence reading• Full sequence information rarely obtained • You need to know the genomic sequence of organism of your

interest

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Desired machine characteristics• Sensitive

• Acquire low abudance peptides• Short acquistion times for high abudance peptides

• Fast• More spectras = more identifications

• Acurate• For effective data processing and confidence of your IDs

• High resolving power• Resolve nearly isobaric precursors

• Integration of more fragmentation approaches• ETD for PTM analysis

• Allow higher order of MSn fragmentation• Presence of ion trap• Perform reporter ion quantification in MS3

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Hybrid instruments• Each type of mass analyser has disadvantages• Overcome by combination of analysers into one

machine • Until summer 2013 two was usual combination• Fusion – first tribrid instrument on the market

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Tribrid instrument

Ion trapFast and sensitive detector

OrbitrapPrecise and high resolution detector

Quadrupole filter

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Main advatages• Orbitrap

• Fourier transformation type of mass analyser• High resolution (450 000) and accuracy (sub ppm)

• Qadrupole• Mass filter• Fast and precise precursor selection• Prevent filling of downstream mass analysers with unwanted ions

• Ion trap is most sensitive type of mass analyser• One charge give detectable signal• Combination with Qudrupole for fast ion selection• Trap filled only with desired ions• Faster and more sensitive• Have limited capacity• 20Hz operating frequency (proteomic application)

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Methods of quantification• Majority of methods - only relative quantification• Targeted approach allows to measure exact

concentrations• Four basic principes

• Labeled in MS1

• SILAC – metabolic labeling during cultivation• Dimethyl labeling – labeling after digestion

• Labeled at MS2 level - detection of reporter ions• TMT• ITRAQ

• Label-free• DIA-based methods

• SWATH

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Reporter ions quantification

Quantification in MS2 suffers from ratio flattening

Fusion can select multiple precursors in IT from MS2 , fragment them and measure ratios in MS3

Speed is reduced to one half in comparison with MS2 approach, but results are better

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Post-transcriptional modifications

• We don´t have ETD (electron transfer disociation) • Allows effective study of glycosylations

• Technically we can analyse phosphorylation, ubiquitination and alternative N-terminal sequence

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Quality Control in our facility• Regular check of chromatogram profile, intensity

and number of IDs in our standart sample and in Thermo Hela digest• From 2hours gradient we acquire 4500-5000 IDs with

1%FDR

• Check (minimally twice per day) peak shape and intensity on Cyt C Thermo standart

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Our current offer• Gel-based protein identification

• Simple one by MALDI Tof/Tof (190kč for piece)• Complex one on Fusion

• Complex proteome analysis • 4500-5000 IDs from 120min gradient (2,5 hours of instrument

time) from in house prepared human cell lysate, 4900 IDs from 120min gradient of Thermo Hela digest

• Including sample preparation• Detergent (SDC) lysis• Trypsin cleavage and sample desalting

• Relative quantification of samples (not all IDs are quantified)

• Up to 10 samples with TMT• Unlimited number with label-free approach• SILAC samples (SILAC labeling must be done by

customer)

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Software equipment• Protein identification and TMT quantification

• Proteome discoverer• Scaffold

• Label-free quantification• Max Quant and Perseus • Sieve

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We will offer following services • Multiple reaction monitoring via targeted

approach• Phosphoproteome analysis

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Our prices• We dont want co-authorship of publications, our

services are charged• Prices set to cover staff, maintenance and consumables • Not definitive, will be revisited after 6 months of facility run• Costs of laboratory maintenance (energy, services) is not yet

included

• 750 Kč for one hour of instrument time – including operator´s time and basic data analysis

• 2500 Kč for TMT labeling of one sample• 300 Kč for one hour of operator´s time (data analysis)• 400 Kč sample preparation (FASP detergent method)

• 250kč consumables• 150kč operator´s time

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Examples of current projects• Comparison of five yeast strains, two cells

populations from each. Totaly 10 diffrent samples.• 4000 Kč sample preparation

• Label-free• 33750 Kč measurement of samples (60minutes gradient) in

triplicate• Or TMT

• 27000 Kč TMT labeling• 7500 Kč measurement of sample in duplicate(4 hours

gradient)

Total price of both approaches under 40000 Kč – 4000 Kč for sample

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Pull-down experiments

• Basic quantification based on three best peaks (top three), not really reliable• 800 Kč sample preparation• 3000 Kč for two 60min gradients

• Label-free quantification - reliable• 800 Kč sample preparation• 9000 Kč for 6 1H gradients• 600 Kč data analysis

Laboratory of sequencing DNA Faculty of Science, Charles University of Prague

YEAR Number of Sequencing

Number of Fragment analysis

2009 10 700 8 100

2010 13 300 16 150

2011 20 650 13 050

2012 22 900 13 200

2013 23 500 17 000

2014 26 900 9 500

https://www.natur.cuni.cz/biologie/servisni-laboratore/laborator-sekvenace-dna

Founded in 2003.Present – laboratory is self-sufficient including salaries of two employees.

Application• High-throughput Next-

generation sequencing on MiSeq Illumina

• Sanger sequencing and fragment analysing on

ABI 3500XL genetic analyzer

Sanger sequencing

Wikimedia

Read length - 800 bp

Sanger sequencing• 3500 XL• obrázek

• Template (DNA) PreparationType of DNA templates sequenced - PCR product, Plasmid, Phage/cosmid, BAC clones etc. After generation, PCR products need to be purified to remove excess primers and 4 dNTPs. Purification can be done either enzymatically, or by passing through a column or from a gel. Other templates, e.g. plasmids are usually prepared in sequence quality grade.

• Cycle Sequencing Reaction by Sanger's dideoxy Terminator Method on a PCR MachineComponents - DNA, primer, heat resistant DNA polymerase, 4 dNTPs, 4 dideoxy terminator nucleotides (4 ddNTPs) fluorescently labeled with 4 different dyes, and enzyme buffer containing Mg++, K+.

• Fragment Separation by Capillary Electrophoresis on ABI 96-capillary 3730XL SequencerThe samples are electrokinetically injected into the array of capillaries, The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragment's base sequence. A laser beam excites these dye molecules as the fragments reach a detection window producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD camera. Very sophisticated optical and electronic devices produce a color readout that is translated, with the help of a sequence analysis software, into a sequence, as we see it.

Fragment analysis

Microsatellite analysis AFLP, RFLP SNP genotyping

Calibration for 4-dye, 5-dye and 6-dye samples:

DS-30 (6FAM™, HEX, NED™, ROX™) - standard ROX 400, 500

DS-33 (6FAM™, VIC®, NED™, PET®, LIZ®) - standard LIZ 500, 600

DS-02 (dR110, dR6G, dTAMRA™, dROX™, LIZ®) - standard LIZ 120

DS-36 (6FAM™, VIC®, NED™,SID™,TAZ™,LIZ®) - standard LIZ 500,

600

Next generation sequencing• Amplicon sequencing• 16S Metagenomics• Small genome de novo sequencing• Small genome reseq. – indel, SNPs• RNA sequencing – gene expresion, transcriptom

sequencing, smallRNA seq.• Sequencing library quality control

Next generation sequencing

Other equipmentThe 2100 Bioanalyzer Agilent is a microfluidics-based platform that is used for the quantification and qualification analysis of DNA, RNA, proteins, and cells.evaluation of RNA, DNA, and protein samples

The Covaris M220 Focused-ultrasonicator generate fragment sizes between 150 and 5,000 bpin tight, highly reproducible fragment distributions and is designed for next generation sequencing applications that require high-quality DNA fragmentation for library preparation.

Quantification: Accurate quantification of input material is crucial to efficient library construction and sequencing. Quantus Fluorometer, NanoDrop Spectrophotometer