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.

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Yu LR, Zhu Z, Chan KC, Issaq HJ, Dimitrov DS, Veenstra TD

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.

J Proteome Res. 2007 Nov;6(11):4150-62. Epub 2007 Oct 9.

PubMed ID
17924679 [ View in PubMed
]
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.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Retinoic acid-induced protein 3Q8NFJ5Details
DNA topoisomerase 2-alphaP11388Details
Multifunctional protein ADE2P22234Details
Ribonucleoside-diphosphate reductase subunit M2P31350Details
Heat shock protein beta-1P04792Details
Ribosome-binding protein 1Q9P2E9Details
Microtubule-associated protein 4P27816Details
Dual specificity protein kinase TTKP33981Details
Triosephosphate isomeraseP60174Details
Phosphoglucomutase-1P36871Details
VimentinP08670Details
Spectrin beta chain, non-erythrocytic 1Q01082Details
Membrane-associated progesterone receptor component 1O00264Details
Nucleolar and coiled-body phosphoprotein 1Q14978Details
Heterogeneous nuclear ribonucleoproteins A2/B1P22626Details
Heterogeneous nuclear ribonucleoprotein KP61978Details
Filamin-AP21333Details
NucleophosminP06748Details
60S ribosomal protein L14P50914Details
ZyxinQ15942Details
Splicing factor 3B subunit 1O75533Details
Serine/threonine-protein kinase N2Q16513Details
ATP-citrate synthaseP53396Details
Proliferation marker protein Ki-67P46013Details
ELAV-like protein 1Q15717Details