Journal
PROTEOMICS
Volume 6, Issue 2, Pages 571-578Publisher
WILEY
DOI: 10.1002/pmic.200500267
Keywords
immunoprecipitation; MS; phosphopeptide purification; tyrosine phosphorylation
Funding
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR017990] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R21NS044184] Funding Source: NIH RePORTER
- NCRR NIH HHS [S10 RR017 990-01] Funding Source: Medline
- NINDS NIH HHS [R21 NS44184-01] Funding Source: Medline
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Identification of tyrosine phosphorylation by MS is challenging due to its low abundance in biological samples. Therefore, specific enrichment of tyrosine phosphorylated peptides prior to their analysis is highly desirable. The application of immunopurification of phosphotyrosine (pY) peptides using pY antibodies has been greatly limited by poor selectivity. In the present study, we have shown that the selectivity of pY peptide immunopurification can be dramatically improved by adding detergents to immunoprecipitation buffers. Optimum selectivity and sensitivity were achieved using an immunoprecipitation buffer containing n-octyl glucoside with a concentration above its critical micelle concentration (0.7%). The optimized method was used to identify in vivo tyrosine phosphorylation on proteins isolated from cell extract by anti-pY protein immunoprecipitation. After immunopurification, non-pY-containing peptides from protein digests were readily removed and pY peptides became the dominant peaks in MALDI quadrupole-TOF mass spectra. In addition, the signal intensities from pY-containing peptides were enhanced significantly after enrichment, allowing characterization of tyrosine phosphorylation sites with greater sensitivity.
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