Pharmaceutical Sciences Faculty Publications

Improved Titanium Dioxide Enrichment of Phosphopeptides from HeLa Cells and High Confident Phosphopeptide Identification by Cross-Validation of MS/MS and MS/MS/MS Spectra

Document Type

Article

Publication Date

11-1-2007

Journal Title

Journal of Proteome Research

ISSN

1535-3893

Volume

6

Issue

11

First Page

4150

Last Page

4162

DOI

10.1021/pr070152u

PubMed ID

17924679

Abstract

Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate.

Keywords

Caseins, cattle, chromatography, high pressure liquid, gene expression regulation, neoplastic, glutamic acid, mass spectrometry, peptides, phosphopeptides, proteomics, titanium, trypsin

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