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prca1550-sup-0001-FigureS1.docx5824K

Figure S1. A. Anti-Clathrin western blot in exosomal and crude human plasma protein extracts (exosome fractions from human plasma, n=6; crude human plasma fractions, n=3; pValue from Student tTest<0.05). B. Subcellular distribution of total protein identifications in both exosome and microparticle (MP) fractions, as provided by String software (http://string-db.org/).

Figure S2. MS data monitoring during both LC-MS sets (exosome extracts, A; microparticles fractions, B). Intensity and retention time are checked for two standard cytochrome-C peptides (KYIPGTK, m/z 403.7422 and MIFAGIK, m/z 390.2278).

Figure S3. Overlaid basepeak chromatograms of triplicate LC-MS runs (black-green-blue) for all the analyzed samples over the exosome and microparticle acquisition sequences.

Figure S4. Distribution of precursor ions intensity for the different analytical runs of the exosome or microparticle series, shown as histograms or box-plot representations. Some raw files showing very low intensity signal, corresponding to particular replicate injections (one MS replicate of sample 9A in the exosome series, and one MS replicate of samples 7A and 10A in the microparticles series) were rejected for further quantitative analysis.

Figure S5. Distribution of the CVs across technical replicate MS analysis for the entire population of proteins quantified in each clinical sample, after processing with MaxQuant or MFPaQ. In total, 20 and 16 samples were analyzed for the exosome and microparticle series respectively, for which triplicate LC-MS runs were obtained (except for 5 samples for which only two replicate runs were available). For each sample and each protein, the CV across technical replicates was calculated on quantifiable proteins (see Supplementary Methods), after imputation of missing values, using the quantitative metric generated by the two software (LFQ or PAI), and the figure shows the boxplot of the distribution of these CV for the entire population of proteins quantified in each sample. Lower and upper limits of the box: 1rst and 3rd quartile of the population, inner black bar: median, red cross: mean, whiskers:5- and 95-percentile, blue marks: min and max values. The size of the population (total number of quantified proteins) for each quantitative analysis is indicated on each graph.

Figure S6. Evaluation of the repeatability between technical replicates using MaxQuant or MFPaQ quantification. For each sample, the ratio between replicate runs was calculated for each protein (for samples analyzed in triplicate runs, 3 different ratios were obtained), and in each binary comparison, the percentage of proteins found to be regulated using 2 time cutoff was plotted.

Figure S7. Different sources of variability in the analytical workflow. A. Evaluation of the repeatability of the protocol for microvesicles isolation in an independent test experiment, using a test plasma sample divided in identical 2mL aliquots. Triplicate exosome sample preparation were performed and analyzed by LC-MS, the graph shows the box-plot of proteins CV distribution across these different sample preparations, with a median protein CV around 20%. CVs are calculated using the MFPaQ PAI metric. B. Evaluation of the MS technical variability during the analysis or the clinical samples: box-plots show the distribution of the CVs across triplicate MS analysis for the entire population of exosomal proteins quantified in each clinical sample, after processing with MFPaQ. C. Biological variability associated to the different exosome samples in AAA or control clinical groups. CVs were calculated from the averaged PAI value (from technical replicates), across all the samples of each group of the study.

prca1550-sup-0002-TableS1.xlsx3245KTable S1
prca1550-sup-0003-TableS2.xlsx1185KTable S2

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