title: Quantitative proteomics using ultralow flow capillary electrophoresis-mass spectrometry.
abstract: In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE-MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE-MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE-MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE-MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE-MS analysis.
authors.affiliation: †Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Faserl
authors.affiliation: †Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Leopold
authors.initials: L
authors.lastname: Kremser
authors.affiliation: ‡Division of Cell Biology, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Müller
authors.affiliation: ‡Division of Cell Biology, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: David
authors.initials: D
authors.lastname: Teis
authors.affiliation: †Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Lindner
mesh_terms: D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics
chemical_list: D010455:Peptides; D011506:Proteins; D020543:Proteome
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