title: Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes.
abstract: The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high-performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600-min gradient, with average chromatographic peak widths of less than 1 min. At room temperature, a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02215-5450, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhou
authors.forename: Yu
authors.initials: Y
authors.lastname: Lu
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: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D051379:Mice; D008969:Molecular Sequence Data; D011312:Pressure; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D013696:Temperature
chemical_list: D020543:Proteome