High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS.
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.
Berger
Scott J
SJ
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Lee
Sang-Won
SW
Anderson
Gordon A
GA
Pasa-Tolić
Lijana
L
Tolić
Nikola
N
Shen
Yufeng
Y
Zhao
Rui
R
Smith
Richard D
RD
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
D000596:Amino Acids; D010455:Peptides; D011492:Protein Hydrolysates; D010450:Endopeptidases