Enrichment of Phosphopeptides by IMAC

Phosphopeptides can be specifically enriched by immobilized metal affinity chromatography (IMAC), which utilizes the interaction of chelator-immobilized three-valent cations such as Fe3+ or Ga3+ with the phosphate group [30-32]. The efficiency of this approach is shown in Figure 6.12, which shows the results of nanoESI-MS analysis

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Fig. 6.12 Enrichment of phosphopeptides using IMAC. (a) NanoESI-MS survey spectrum of an elastase digest of protein kinase A. (b) The same sample and MS analysis as in (a) but after enrichment of phosphopeptides by IMAC. All signals labelled with 'P' were found to represent phosphopeptides upon subsequent product ion analysis. [33].

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Fig. 6.12 Enrichment of phosphopeptides using IMAC. (a) NanoESI-MS survey spectrum of an elastase digest of protein kinase A. (b) The same sample and MS analysis as in (a) but after enrichment of phosphopeptides by IMAC. All signals labelled with 'P' were found to represent phosphopeptides upon subsequent product ion analysis. [33].

of an elastase digest of protein kinase A before and after enrichment of phosphopep-tides by IMAC.

As can be seen by comparing Figure 6.12 a and b, the complexity of the peptide mixture is greatly reduced by the IMAC step, and most signals in the spectrum after IMAC (Fig. 5.12 b) were identified as phosphopeptides. The method also tends to enrich peptides having Wor H residues or having (multiple) residues of D and E, since the sidechains of these amino acids also interact with free coordination sites of immobilized metal cations. Recently, methyl ester formation of free carboxyl groups was introduced to suppress the nonspecific interaction of D and E residues [34], and the efficiency of this measure was demonstrated in an application to the yeast proteome. Phosphopeptide enrichment by intermediate covalent binding to a solid support via a phosphoamide bond and subsequent release has also been demonstrated [35].

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