Sequence-Dependent Enrichment of a Model Phosphopeptide: A Combined MALDI-TOF and NMR Study

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• 作者：Lucre虁ce Matheron ; Emmanuelle Sachon ; Fabienne Burlina ; Sandrine Sagan ; Olivier Lequin ; Ge虂rard Bolbach
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：April 15, 2011
• 年：2011
• 卷：83
• 期：8
• 页码：3003-3010
• 全文大小：912K
• 年卷期：v.83,no.8(April 15, 2011)
• ISSN：1520-6882

文摘

The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO₂ or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH₂ in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO₂ affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO₂ was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P⁹鈭?S¹⁰ segment, with formation of a salt bridge between the guanidinium group of the R⁷ side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G₄} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO₂. To improve TiO₂ affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO₂ became possible.