title: Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms.
abstract: The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.
authors.affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215-5450, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhou
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Sikorski
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.forename: James T
authors.initials: JT
authors.lastname: Webber
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin