ArticleAnalytical biochemistry2008
Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution.
Article in Analytical biochemistry, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 46 citations in OpenAlex.
- Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.Chemical communications (Cambridge, England) · 2023Review
- Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.Analytical chemistry · 2023Article
- Exploring protein phosphorylation by combining computational approaches and biochemical methods.Computational and structural biotechnology journal · 2020Article
- Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.Molecular & cellular proteomics : MCP · 2016Article
- Efficient Esterification of Oxidized l-Glutathione and Other Small Peptides.Molecules (Basel, Switzerland) · 2015Article
- Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling.Methods (San Diego, Calif.) · 2013Review
- Protein analysis by shotgun/bottom-up proteomics.Chemical reviews · 2013Review
- C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization.Journal of biomolecular techniques : JBT · 2013Article
- Enrichment techniques employed in phosphoproteomics.Amino acids · 2012Review
- Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.Analytical chemistry · 2011Article
- Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides.Analytical biochemistry · 2011Article
- Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.Journal of the American Society for Mass Spectrometry · 2010Article
- Studies of phosphoproteomic changes induced by nucleophosmin-anaplastic lymphoma kinase (ALK) highlight deregulation of tumor necrosis factor (TNF)/Fas/TNF-related apoptosis-induced ligand signaling pathway in ALK-positive anaplastic large cell lymphoma.Molecular & cellular proteomics : MCP · 2010Article
- Proteomics: challenges, techniques and possibilities to overcome biological sample complexity.Human genomics and proteomics : HGP · 2009Article
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.