HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M600480-MCP200v1 6/8/1380    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Imanishi, S. Y. Articles by Eriksson, J. E. Search for Related Content PubMed PubMed Citation Articles by Imanishi, S. Y. Articles by Eriksson, J. E. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 6:1380-1391, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Reference-facilitated Phosphoproteomics

FAST AND RELIABLE PHOSPHOPEPTIDE VALIDATION BY µLC-ESI-Q-TOF MS/MS^*,S

Susumu Y. Imanishi^,, Vitaly Kochin^,, Saima E. Ferraris^,¶, Aurélie de Thonel^,, Hanna-Mari Pallari^,¶, Garry L. Corthals and John E. Eriksson^,,||

From the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20521 Turku, Finland, Department of Biology, Åbo Akademi University, FIN-20521 Turku, Finland, and ^¶ Department of Biology, University of Turku, FIN-20014 Turku, Finland

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO₂) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO₂ chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. O. Collins, L. Yu, I. Campuzano, S. G. N. Grant, and J. S. Choudhary Phosphoproteomic Analysis of the Mouse Brain Cytosol Reveals a Predominance of Protein Phosphorylation in Regions of Intrinsic Sequence Disorder Mol. Cell. Proteomics, July 1, 2008; 7(7): 1331 - 1348. [Abstract] [Full Text] [PDF]