Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 15, Issue 5, Pages 647-653Publisher
AMER CHEMICAL SOC
DOI: 10.1016/j.jasms.2003.12.019
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Funding
- NIGMS NIH HHS [F32 GM069315, F32 GM069315-01, F32 GM069315-02] Funding Source: Medline
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A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phospliatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chomatography-mass spectrometry (muLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorytated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within similar to20%). (C) 2004 American Society for Mass Spectrometry.
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