HeLa proteome of 12,250 protein-coding genes
We developed an optimized multi-shot proteomics workflow based on high-resolution offline high pH reversed-phase peptide separation of high peptide loads collecting many fractions that were in turn analyzed by short online chromatographic separations and fast peptide sequencing using orbitrap tandem mass spectrometry.
Sample Processing Protocol
Protein concentration was estimated by Bradford assay (Bio-Rad), and the lysates were digested with Lys-C (Wako) in an enzyme/protein ratio of 1:100 (w/w) for 1 h followed by a threefold dilution with 25 mM Tris, pH 8.5, to 2 M GndCl and further digested overnight with trypsin (Sigma Aldrich) 1:100 (w/w). For experiments using different proteases, lysates were directly diluted to 2 M GndCl before addition of proteases (Lys-C, Trypsin, Chymotrypsin (Roche) and Glu-C (Roche)). Protease activity was quenched by acidification with trifluoroacetic acid (TFA) to a final concentration of approximately 1%, and the resulting peptide mixture was concentrated using reversed-phase Sep-Pak C18 Cartridge (Waters). Peptides were eluted off the Sep-Pak with 2 mL 40v% acetonitrile (ACN) followed by 2 mL 60% ACN. The ACN was removed by vacuum centrifugation for 40 min at 60 °C and the final peptide concentration was estimated by measuring absorbance at 280 nm on a NanoDrop (NanoDrop 2000C, Thermo Scientific).
Data Processing Protocol
All raw LC–MS/MS data were analyzed by MaxQuant v220.127.116.11 using the Andromeda Search engine and searched against the complete human Uniprot database including all Swiss-Prot and TrEMBL entries as well as all isoforms. Four analysis groups were made in MaxQuant, enabling one combined analysis for all proteases. Carbamidomethylation of cysteine was specified as fixed modification for all groups. Variable modifications considered were oxidation of methionine, protein N-terminal acetylation, pyro-glutamate formation from glutamine and phosphorylation of serine, threonine, and tyrosine residues. The match between run feature and the second peptide option was disabled, and everything else was set to the default values, including the false discovery rate limit of 1% on both the peptide and protein levels. For PTM analysis, the HeLa dataset was searched separately using methylation of arginines and lysines or acetylation of lysines as variable modifications.
Bekker-Jensen DB, Kelstrup CD, Batth TS, Larsen SC, Haldrup C, Bramsen JB, Sørensen KD, Høyer S, Ørntoft TF, Andersen CL, Nielsen ML, Olsen JV. An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes. Cell Syst. 2017 Jun 6. pii: S2405-4712(17)30188-6 PubMed: 28601559