Progenesis LC-MS Differential Expression Analysis Workflow...
Transcript of Progenesis LC-MS Differential Expression Analysis Workflow...
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Progenesis LC‐MS ‐ Differential Expression Analysis Workflow Guide This guide shows how you to quickly and easily analyse label‐free LC‐MS/MS data to measure differential expression and identify proteins of interest using Progenesis LC‐MS. To try this analysis workflow you can use the tutorial data and a Help document (PDF) included within the software alongside this guide. The tutorial compares protein expression differences between two strains of C. Difficile* using 2 groups of 3 technical replicates. We also encourage you to evaluate Progenesis LC‐MS using some of your own data. To request a software demonstration or licence your own LC‐MS runs for analysis please contact [email protected].
*Data courtesy of Dr. Rob Parker and Prof. Haroun Shah, Health Protection Agency, London, UK
Import your data
Tutorial runs are automatically loaded following theImport step above or you can add your own runs inthe following file formats, .mzXML, Waters .RAW,Thermo .RAW and NetCDF. For each run you willsee a 2D image representing multiple MS scanstaken over time.
Select the Reference Run that all other runs arealigned to. It is best to select a Reference Run thatrepresents stable LC‐MS conditions, has minimalnoise and includes all the important features youwant to compare between your different biologicalgroups e.g. control vs. treated.
Select your Reference Run
Align your LC‐MS data
Getting started using the tutorial data‐set. Open Progenesis LC‐MS, select “Download Tutorial” and put thetutorial folder onto your desktop when prompted. Use the browse option, bottom left in the start‐up screen andlocate the tutorial folder. Select the “Tutorial_LC‐MS” file. Create a new experiment using this data by clicking“Import” and follow the steps below. Getting started using your own data. Open Progenesis LC‐MS and select the “New” option in the start‐up
screen. Name your experiment, select the correct data and instrument type for your runs where prompted on‐screen and follow the steps below for analysis.
Alignment is the most important step in theworkflow. It corrects retention time variationbetween runs, ensures 100% matching of peptideions within your LC‐MS data and enables robuststatistical analysis. Apply “Automatic Alignment”with a single click and check the alignment using thevisual tools. Look at the two views on the right handside and if they show little visual disturbance thenalignment has been achieved. This should occur inmost cases. In cases where Automatic Alignment doesn’t workyou can view the runs unaligned, add manualvectors to improve areas that need attention andsee the effects by switching to an aligned view. Theresult of alignment is a single “Aggregate Run”representing all peptide ion data from all samples inthe same coordinate space.
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By clicking section complete the softwareautomatically analyses all the aligned runs. Everypeptide ion in the experiment is detected, normalised and quantified. The result is a completeset of LC‐MS peptide ion outlines represented onthe aggregate run that are applied consistently toall the runs within the experiment.
Detection, Normalisation and Quantification
Set up your experimental
groups
View your results This step automatically displays a list of detectedpeptide ions ranked by ANOVA (p‐value) based ondifferences compared between your groups. Resultscan be validated using the expression profiles, datatables, 1D, 2D and 3D views. This helps youcharacterise the peptide ions displaying interestingbehaviour. You can also see where peptide ions arelocated on your aggregate run image or on anyindividual LC‐MS run images. Peptide ions can beselected and grouped in different ways using tags.Tags are used to create sub‐groups of peptide ionsbased on the characteristics you’re interested in.You can also edit any peptide ions, which onlyneeds to be done on a single run as any edits youmake are propagated across all other runs. At thisstage peptide ions of interest can be taken forwardfor multivariate statistical analysis andidentification. Any peptide ions without associatedMS/MS data can be exported as an inclusion list forfurther LC‐MS/MS runs (see below).
Organise your runs into the groups to match yourexperimental design e.g. Control vs. Treated. Youcan also set up other groupings such as Male vs.Female to measure differential expression betweenthem within the same experiment.
Optional Step. Sort peptide ions using the datatable view by clicking the “ms/ms” column heading.Select any features showing “0”. From the Filemenu select “generate inclusion list” and export thislist as a .txt file. This is used to target peptides forMS/MS in further runs. Once you have additional MS/MS data from newruns you can import this into your experiment (seethe first step in this workflow guide) WITHOUTaffecting the quantification results of your currentdata analysis. You can repeat this process while your LC system isstable and add more data to your experiment forreliable identification of proteins.
Filtering Include or exclude detected peptide ions from youranalysis results based on m/z, time, charge stateand number of isotopes.
Add further MS/MS data to your existing LC‐MS results
Generate inclusion list of peptide ions without MS/MS
data
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Progenesis Stats ‐ PCA
Run a Protein Search
In Progenesis Stats you first see a Principal Components Analysis (PCA) plot to check your runs cluster based on the expected groups. PCA is anunsupervised technique i.e. it does not use any knowledge of the experiment groupings. Eachcoloured point in the PCA plot is a run, and byhovering over it you can easily indentify it. If one of the runs was an obvious outlier you would want toinvestigate this further. You can filter and tag peptide ions based on their q‐value, used to report false discovery rates and/or their statistical power within the experimental analysis.
View MS/MS data for any interesting peptide ionsyou bring forward from View Results ProgenesisStats. A visual display of the peptide ion shows a redtarget circle to indicate where MS/MS wastriggered. Select the MS spectra you want toperform searches with and export to search againstyour chosen database using Phenyx®, Mascot™ orSEQUEST® search engines.
You can “Ask another question” and group peptideions according to their expression profiles usingCorrelation Analysis. You can look at all of thepeptide ions in this way or use tags to select a sub‐set of peptide ions to display based on user‐definedcharacteristics. The dendrogram is interactive soyou can click, select and zoom‐in on different areas to find similar expression patterns. You can alsodrag the grey sliding bar on the dendrogram tohighlight the different, colour coded, expressiongroups.
Import your Protein Search
results
Progenesis Stats – Ask more questions?
Optional step. You can switch between viewingyour data in Progenesis Stats and the View Results step. It is easy to navigate between the two stepsusing the trail of icons at the top of the software.You can apply tags to create a sub‐set of peptide ions based on expression profile (using CorrelationAnalysis), p‐value, q‐value, power or fold‐change to reduce the amount of data that you need to investigate by switching between Progenesis Stats and the View Results step.
Import the search result files that you generate and they are automatically associated with the peptideions that generated the protein identifications. You can filter out any search results based on peptide score or number of peptides. So for example you can set the threshold at 1 to only take forward proteins indentified by two or morepeptide ions. This can be used to increase confidence in results you see at the protein level in the Protein View.
View your results
Progenesis Stats ‐ PCA
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Report
At this final step you can create a report of your quantitative and qualitative analysis results. Itprovides an easy way to share results with yourcolleagues and can be customised to include arange of characteristics selected from acomprehensive list. You can also produce a targeted report based on the sub‐set peptide ions you have tagged as most interesting.
Resolve peptide ions associated with different
proteins
Protein View ‐Expression differences
Moving into this penultimate step automatically brings together quantified LC‐MS data and qualitative LC‐M/MS results to define the proteinsof interest within your experiment. You can alsoexplore individual peptide ions associated with eachidentified protein. The peptide ions are used to calculate fold change with ANOVA (p‐value) for the proteins they areassociated with. Identified proteins can be rankedand tagged based on tabled information to generate a targeted list of differentially expressedproteins.
You may see one or more “conflicts” in the table ofresults, top right. This means that a peptide ion matches a sequence from more than one identifiedprotein. This highlights cases where it is ambiguous which protein is differentially expressed within theexperiment. The most likely protein identity, based on highest protein score is shown in the top left hand table.Protein score is calculated from the individualpeptide ion scores that combine to give the proteinidentification. By clicking the Conflict Resolution tabyou can view all the other protein identifications(bottom left) attributed to the peptide ion. You can resolve ambiguous results by either reassigning the peptide ion to an alternative protein identificationor disassociating it from the alternativeidentifications. You may also decide that a result isso ambiguous that you remove the protein and allassociated peptide ions from being considered assignificant. You will notice as you add or remove peptide ionsassociated with a protein identity that the proteinabundance, score, no. of peptides and ANOVA p‐value change to reflect what you’re doing.
Congratulations this completes the data analysis workflow to quantify differentially expressed peptide ions and proteins, generate inclusion lists and add additional MS/MS data to an existing experiment.
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For more information on analysis workflows and to arrange a demo on
your own data visit, www.nonlinear.com/LC‐MS