title: High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS.
abstract: In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Berger
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Gordon A
authors.initials: GA
authors.lastname: Anderson
authors.forename: Lijana
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: Nikola
authors.initials: N
authors.lastname: Tolić
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Shen
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D020374:Chromatography, Micellar Electrokinetic Capillary; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D016208:Databases, Factual; D010450:Endopeptidases / Q000737:chemistry; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D011492:Protein Hydrolysates / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry
chemical_list: D000596:Amino Acids; D010455:Peptides; D011492:Protein Hydrolysates; D010450:Endopeptidases