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In JoVE (1)
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Articles by Brian D. Steinert in JoVE
Brian D. Steinert, Shijia Zhao, Linxia Gu
Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln
Other articles by Brian D. Steinert on PubMed
Highly Specific Capture and Direct MALDI MS Analysis of Phosphopeptides by Zirconium Phosphonate on Self-assembled Monolayers
Analytical Chemistry. Jan, 2010 | Pubmed ID: 19968246
The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO(2) and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility. In this report, we present the first demonstration of the use of immobilized zirconium on a phosphonate-terminated self-assembled monolayer (SAM) for specific phosphopeptide capture and direct analysis by MALDI MS. By using the herein described functionalized-surface-based technology, efficient enrichment of phosphopeptides in different standard test systems such as alpha- or beta-casein digests or synthetic phosphopeptides spiked in nonphosphorylated protein digest has been demonstrated. The limit of detection for a beta-casein phosphopeptide was assessed to be at the low femtomole level. Compared to other state-of the-art technologies, like use of TiO(2) and Fe-IMAC, the presented technique demonstrated a superior performance with respect to specificity and bias with respect to singly or multiply phosphorylated peptides. Additionally, this platform was also successfully applied for ESI sample preparation, providing detailed sequence information of the investigated phosphopeptide. This technology was also proven to be applicable for real life samples such as phosphorylation site analysis of recombinant human MAPK1 and HSP B1 isolated from a 2D-gel spot by phosphopeptide enrichment and direct MALDI MS/MS.