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nmrlearner 03-25-2011 06:34 PM

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

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

Anal Chem. 2011 Mar 23;

Authors: Lucre?ce M, Emmanuelle S, Fabienne B, Sandrine S, Olivier L, Ge?rard B

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 TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible.

PMID: 21428305 [PubMed - as supplied by publisher]



Source: PubMed


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