Specific Phosphopeptide Enrichment with Immobilized Titanium Ion Affinity Chromatography Adsorbent for Phosphoproteome Analysis

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• 作者：Houjiang Zhou ; Mingliang Ye ; Jing Dong ; Guanghui Han ; Xinning Jiang ; Renan Wu ; Hanfa Zou
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：September 5, 2008
• 年：2008
• 卷：7
• 期：9
• 页码：3957-3967
• 全文大小：322K
• 年卷期：v.7,no.9(September 5, 2008)
• ISSN：1535-3907

文摘

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti⁴⁺-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti⁴⁺ through the chelating interaction between phosphate groups on the polymer and Ti⁴⁺. The resulting Ti⁴⁺-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti⁴⁺-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti⁴⁺-IMAC to other enrichment methods including Fe³⁺-IMAC, Zr⁴⁺-IMAC, TiO₂ and ZrO₂, and demonstrate superior selectivity and efficiency of Ti⁴⁺-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti⁴⁺-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.