Computational protocol: Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals

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[…] Raw spectral data from Xcalibur™ (Thermo Scientific, v2.1) was searched against a custom database compiled with (a) the RefSeq human reference proteome with isoforms (88,354 entries, downloaded from www.uniprot.org on 18th July 2013), (b) the sequence for trypsin from Sus scrofa (accession P00761 from www.uniprot.org), (c) the predicted proteome of Plasmodium vivax (5,393 entries, downloaded from www.plasmodb.org release 9.2), (d) a subset of 183 reannotated Plasmodium vivax proteins, totalizing 93,931 entries. The database containing P. vivax sequences is used routinely in our laboratory, and this study, involving healthy individuals, represents an internal control of an unrelated species, helping set an appropriate level of parameter stringency. The search was done with the Sequest HT algorithm on the Proteome Discoverer™ Software version 1.4.1.14 (Thermo Scientific). Searches were performed with the following parameters: digestion by trypsin, 2 missed cleavage sites allowed, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.6 Da, oxidation of methionine as the variable modification and carbamidomethylation of cysteine as the fixed modification. The signal-to-noise (S/N) threshold was set to 1.5. Percolator was used for PSM validation at 1% false discovery rate (FDR) at peptide level. High confidence peptides (1% FDR) were further filtered at ΔCn ≤0.1 and Xcorr greater than 1.5, 2.0, 2.25, 2.5, 2.75, 3.0, 3.2 and 3.4 for charge states 1, 2, 3, 4, 5, 6, 7 and >7, respectively. Proteins identified by the same set of peptides were grouped under one master protein entry. The normalized spectrum abundance factor (NSAF) of proteins within a sample was calculated as previously described (). Finally, proteins identified with 2 or more peptides were automatically considered in the final list, and in the cases of proteins of interest identified by single peptides, their spectra were manually annotated to ensure a confident identification. […]

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