A novel quantitative proteomics strategy to study phosphorylation-dependent peptide-protein interactions.
Phosphorylation-dependent protein-protein interactions provide the mechanism for a large number of intracellular signal transduction pathways. One of the goals of signal transduction research is to understand more precisely the nature of these phosphorylation-dependent interactions. Here, we report a novel strategy based on quantitative proteomics that allows for the rapid analysis of peptide-protein interactions with more than one phosphorylation site involved. The phosphorylation of two tyrosine residues, Y342 and Y346, within the linker B region of the protein-tyrosine kinase Syk is important for optimal signaling from the B cell receptor for antigen. We employed four amino-specific, isobaric reagents to differentially label proteins interacting in vitro with four Syk peptides containing none, one, or two phosphates on tyrosine residues Y342 and Y346, respectively. In total, 76 proteins were identified and quantified, 11 of which were dependent on the phosphorylation of individual tyrosine residues. One of the proteins, peroxiredoxin 1, preferably bound to phosphorylated Y346, which was further verified by Western blotting results. Thus, we demonstrate that the use of 4-fold multiplexing allows for relative protein measurements simultaneously for the identification of interacting proteins dependent on the phosphorylation of specific residues.
Zhou
Fei
F
Department of Medicinal Chemistry and Molecular Pharmacology, and Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, USA.
Galan
Jacob
J
Geahlen
Robert L
RL
Tao
W Andy
WA
D000465:Algorithms; D001665:Binding Sites; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D054464:Peroxiredoxins; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000072377:Syk Kinase
D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D010544:Peroxidases; C515940:PRDX1 protein, human; D054464:Peroxiredoxins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase