title: Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides.
abstract: We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (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 69Ga-71Ga 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.
authors.affiliation: Department of Chemistry and Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Grady R
authors.initials: GR
authors.lastname: Blacken
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
authors.forename: Frantisek
authors.initials: F
authors.lastname: Turecek
mesh_terms: D000595:Amino Acid Sequence; D004952:Esters / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D015394:Molecular Structure; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004952:Esters; D007477:Ions; D008670:Metals; D010748:Phosphopeptides; D012996:Solutions