New approach for analysis of the phosphotyrosine proteome and its application to the chicken B cell line, DT40.
In this study, we have begun to analyze phosphotyrosyl and associated proteins present in a DT40 chicken B cell line overexpressing the nonreceptor protein-tyrosine kinase, Syk. An anti-phosphotyrosine antibody was used to select tyrosine-phosphorylated proteins. After tryptic digestion, peptides were subjected to a beta-elimination reaction and phosphotyrosine-containing peptides were enriched via immobilized metal affinity chromatography. Several known substrates and candidate substrates for Syk and the location of 22 tyrosine phosphorylation sites were identified.
Zolodz
Melissa D
MD
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA.
Wood
Karl V
KV
Regnier
Fred E
FE
Geahlen
Robert L
RL
D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D000072377:Syk Kinase; D014357:Trypsin / Q000737:chemistry
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D020543:Proteome; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D014357:Trypsin