Interrogating the hidden phosphoproteome.
Postgenomic studies continue to highlight the potential clinical importance of protein phosphorylation signaling pathways in drug discovery. Unfortunately, the dynamic range and variable stoichiometry of protein phosphorylation continues to stymie efforts to achieve comprehensive characterization of the human phosphoproteome. In this study, we develop a complementary, two-stage method for enrichment of cysteine-containing phosphopeptides combined with TMT multiplex labeling for relative quantification. The use of this approach with multidimensional fractionation in mammalian cells yielded more than 7000 unique cys-phosphopeptide sequences, comprising 15-20% novel phosphorylation sites. The use of our approach in combination with pharmacologic inhibitors of the mechanistic target of rapamycin complex 1 and 2 identified several putatively novel protein substrates for the mechanistic target of rapamycin kinase.
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.|Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.
Un-Beom
UB
Kang
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.|Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.
William M
WM
Alexander
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.|Department of Pathology, Dana-Farber Cancer Institute, Boston, MA, USA.|Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.|Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Jarrod A
JA
Marto
D003198:Computer Simulation; D003545:Cysteine / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D058570:TOR Serine-Threonine Kinases; D003545:Cysteine
Cysteine-containing phosphoproteomome; Quantitative proteomics; mTOR; mammalian target of rapamycin complex 1 and 2