title: Selective, sensitive, and rapid phosphopeptide identification in enzymatic digests using ESI-FTICR-MS with infrared multiphoton dissociation.
abstract: Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine alpha- and beta-casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDIYED-pY-YRK, and triphosphorylated TRDI-pY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of th
authors.affiliation: Department of Chemistry, Virginia Commonwealth University, Richmond 23284, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Flora
authors.forename: D C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis; D002417:Cattle; D005583:Fourier Analysis; D007259:Infrared Rays; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D011972:Receptor, Insulin / Q000032:analysis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D011972:Receptor, Insulin