Perseus /R exercise Dataset 1 - PowerPoint Presentation

1. Perseus/R exerciseDataset 1 : used for demo of Perseus interface/functionsFilename : «Ecoli-4prot-MaxQuant.txt» ; this is a label-free (LFQ) dataset of an E.coli total digest spiked with some standard proteins in variable amounts.Dataset 2 : exerciseThis is a SILAC dataset resulting from measurement of total proteome of Jurkat T-cells treated for 20h with an inhibitor of the Hsp90 chaperone. (Fierro-Monti, et al (2013) PloS One, 8(11), e80425.)Filename : «proteinGroups_exp14_M1.txt»; table contains 3 replicates with both raw and normalized (by MaxQuant) H/L ratios. Each protein group has columns listing associated biological annotation (GO terms, KEGG, …).Filename : «proteinGroups_R_exp14_M1.txt»; this table version should be used for the R version of the exercise. R scripts are available either as text format in the «analyze_silac_MQ.html» file or as R file (.Rmd) in «analyze_silac_MQ.Rmd». These R scripts follow the Perseus workflows followed in the next slides, excepted for the last part (ex. 8).

2. Perseus workflow - 11) LOAD MATRIX M1Generic matrix upload (green arrow in the upper left corner); load all columns as preconfigured ; click OK.2) FILTER Filter rows -> Filter rows based on categorical columns -> remove ReverseFilter rows -> Filter rows based on categorical columns -> remove ContaminantsFilter rows -> Filter rows based on categorical columns -> remove Contaminants 2=> 4989 protein groups left.3) ANALYSIS 13.1)Visualization --> histogram of all main columns (all “Ratio H/L….” columns) ; click on "tools" icon on the upper left of the histogram window; change max value to 4.0 => look at distributions:=> H/L ratios should ideally be around 1.0 but are not => normalized ratios are at 1.0.3.2)Basic --> summary statistics (columns) : calculate summary statistics and look at median /average of all columns.3.3) Visualization --> scatter plot (columns): select to display "id" and "Ratio H/L exp14 rep1 20h" (or any other ratio column) note distribution of ratios ; is it easy to evaluate the data with ratios in linear form?

3. Perseus workflow - 2 4) LOG AND SELECT NORMALIZED COLUMNS4.1) Basic-> transform -> log2(x) of all main columns ; click OK4.2) Rearrange -> reorder/remove columns ; remove (send them to the left )the raw, non normalized ratio columns. Only keep the 3 normalized ratios columns ; click OK.5) ANALYSIS 25.1) Visualization --> scatter plot (columns) of "id" and replicate 1; on the left select "tools" icon ; add horizontal/vertical zero lines on the left select magnifying lens tool and activate "rectangular selection" above the "points" panel on the right window, select "Gene names" in the pulldown menu control+click/drag in the plot to select points above/below the main the data cloud; change their color using the rainbow wheel on the right switch to display other replicates; check to see if the proteins selected previously are reproducibly increasing/decreasing in the treated sample (H) relative to the untreated (L). 5.2) Visualization --> multiscatter plot (default parameters ; click OK): in multiscatter plot pulldown menu select "Pearson correlation" and display values; use the Acrobat icon to save the image of the plot.6) MORE FILTERING6.1) Filter rows -> filter based on numerical/main column -> filter for x>2 peptides (=> 4369 protein groups left).6.2) Re-do the multiscatter plot as before => Save plot. Compare multiscatter plots; What change do you observe in the Pearson correlation values ? ******************** “proteinGroups_exp14_M2.txt” contains all changes to this point.

4. Perseus workflow - 3 7) STATISTICAL TESTS 7.1) Tests-> One sample tests -> T-test (default parameters, includes FDR filtering); specify the suffix "FDR" in the box at the bottom; click OK 7.2) How many proteins pass T-test+FDR (column "T-test significant") (=> 339)7.3) Repeat T-test without Benjamini-Hochberg FDR filtering: select "p-value" in the pull down box "Use for truncation" type "pval" in the "suffix" box; click OK.How many proteins pass T-test with p-value at 0.05 and without FDR and? (=> 1576)************* “proteinGroups_exp14_M3.txt” contain everything up to here.8) BIOLOGICAL ANNOTATION Do statistical test on GO and other annotations to see if any is enriched in the proteins changing in the experiment: 8.1) Annot. columns -> Fisher exact test -> select column "T-test significant FDR" ; click OKComment on results , i.e. "Category value“************* “GO-term_result_FDR.txt” contains results of this step.8.2) Find out which proteins have the enriched annotation : go back to data matrix filter rows based on categorical columns --> select annotation of interest; select Mode-> keep matching rows; select Filter Mode-> reduce matrix ; click OK; you can save the obtained table : Menu bar on top right  Generic Matrix export ; save where desired as .txt.