title: Rapid and deep human proteome analysis by single-dimension shotgun proteomics.
abstract: Multiparameter optimization of an LC-MS/MS shotgun proteomics experiment was performed without any hardware or software modification of the commercial instrument. Under the optimized experimental conditions, with a 50-cm-long separation column and a 4-h LC-MS run (including a 3-h optimized gradient), 4,825 protein groups and 37,550 peptides were identified in a single run and 5,354 protein groups and 56,390 peptides in a triplicate analysis of the A375 human cell line, for approximately 50% coverage of the expressed proteome. The major steps enabling such performance included optimization of the cell lysis and protein extraction, digestion of even insoluble cell debris, tailoring the LC gradient profile, and choosing the optimal dynamic exclusion window in data-dependent MS/MS, as well as the optimal m/z scan window.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden;
authors.forename: Mohammad
authors.initials: M
authors.lastname: Pirmoradian
authors.forename: Harshavardhan
authors.initials: H
authors.lastname: Budamgunta
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chingin
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Juan
authors.initials: J
authors.lastname: Astorga-Wells
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
mesh_terms: D002021:Buffers; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
chemical_list: D002021:Buffers; D020543:Proteome