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Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides

Analytical Chemistry, ISSN: 0003-2700, Vol: 78, Issue: 17, Page: 6065-6073
2006
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We report a new method called metal affinity capture that when coupled with tandem mass spectromery (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga or Fe and N,N-bis(carboxymethyl)lysme (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H) ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH < 5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the Ga-Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations. © 2006 American Chemical Society.

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