This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS-MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora-A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC). During the course of this work, we also developed and implemented a robust method for calculation of signal to noise ratio in mass spectra. A manuscript describing this work has been published: Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting. Woo EM, Fenyo D, Kwok BH, Funabiki H, Chait BT. Anal Chem. 2008 80(7):2419-25.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-36
Application #
7954124
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2009-03-01
Project End
2010-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
36
Fiscal Year
2009
Total Cost
$11,861
Indirect Cost
Name
Rockefeller University
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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