Computational protocol: Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation*

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Protocol publication

[…] The raw data acquired were processed with the MaxQuant software version 1.1.0.39 according to the standard workflow (). Database search was performed in MaxQuant with the Andromeda search engine () against International Protein Index Human version 3.68 database (87,083 entries in the forward database, including common contaminants) or against the translation of all ORFs in SGD (Saccharomyces Genome Database, 6752 entries in the forward database, including common contaminants) version from 5 Jan 2010, with initial precursor mass tolerance of 7 ppm and fragment mass deviation of 0.5 Da. The search included cysteine carbamidomethylation as a fixed modification and N-acetylation of protein and oxidation of methionine as variable modifications. Up to two missed cleavages were allowed for protease digestion. For trypsin digested proteins, peptides had to be fully tryptic and for LysC, the peptides had to fully match LysC digestion specificity. The “identify” module in MaxQuant was used to filter identifications at 1% false discovery rate on the peptide and protein level using a reverse database in which the lysines and arginines were swapped with the preceding amino acid (). Only peptides with minimum six amino acid length were considered for identification. For SILAC analysis, two ratio counts were set as a minimum for quantification. The lists of identified proteins were filtered to eliminate reverse hits and known contaminants. Inspection of peptide scores revealed that the minimum Andromeda score in the human nonlabeled cell line data was 61 (Note that Andromeda scores are on average threefold higher than Mascot scores). […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Databases SGD IPI
Application MS-based untargeted proteomics
Organisms Saccharomyces cerevisiae, Homo sapiens
Diseases Multiple Sclerosis