Mass spectrometry based draft of the human proteome
This PXD project contains two projects published on ProteomicsDB (https://www.proteomicsDB.org) as integral part of the publication. The first project entitled 'human body map' (https://www.proteomicsdb.org/#projects/42) involves the analysis of 36 different human tissues and body fluids. The second project entitled 'Cellzome adopted' includes a collection of raw files which comprises identifications of 'missing proteins'.
Sample Processing Protocol
Human tissue specimens were obtained from the bio bank of the TU München following approval of the study by the local ethics committee. Samples were collected within the first 30 minutes after resection, macroscopically resected by an experienced pathologist, snap frozen and stored in liquid nitrogen until use. Body fluids requiring no invasive procedures were provided by volunteers. Proteins were extracted under denaturing conditions and either separated by LDS-PAGE followed by in-gel protease digestion or digested in solution in the presence of chaotropic agents. Peptides were separated by ultra-high pressure nanoscale liquid chromatography on 75 μm x 40 cm reversed-phase columns using gradients of 2-32% acetonitrile in 0.1% formic acid, 5% DMSO. The LC was coupled directly to an Orbitrap mass spectrometer, operating in data dependent mode and using either resonance-type or beam-type collision induced dissociation.
Data Processing Protocol
For peptide identification, tandem mass spectra were processed using Mascot Distiller and searched against Uniprot using Mascot and a target-decoy strategy. In parallel, raw MS files were also processed by MaxQuant/Andromeda. All search results and tandem mass spectra were imported into ProteomicsDB (https://www.proteomicsdb.org) and filtered at 1% PSM FDR and 5% local peptide length dependent FDR.
Wilhelm M, Schlegl J, Hahne H, Moghaddas Gholami A, Lieberenz M, Savitski MM, Ziegler E, Butzmann L, Gessulat S, Marx H, Mathieson T, Lemeer S, Schnatbaum K, Reimer U, Wenschuh H, Mollenhauer M, Slotta-Huspenina J, Boese JH, Bantscheff M, Gerstmair A, Faerber F, Kuster B. Mass-spectrometry-based draft of the human proteome. Nature. 2014 May 29;509(7502):582-7 PubMed: 24870543