title: Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.
abstract: In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795-806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.
authors.affiliation: Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Suman S
authors.initials: SS
authors.lastname: Thakur
authors.forename: Tamar
authors.initials: T
authors.lastname: Geiger
authors.forename: Bhaswati
authors.initials: B
authors.lastname: Chatterjee
authors.forename: Peter
authors.initials: P
authors.lastname: Bandilla
authors.forename: Florian
authors.initials: F
authors.lastname: Fröhlich
authors.forename: Juergen
authors.initials: J
authors.lastname: Cox
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D018929:Cell Culture Techniques; D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D053858:Metabolic Networks and Pathways; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins