title: Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing.
abstract: Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.
authors.affiliation: Endocrinology and Reproduction Research Branch, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Robert A
authors.initials: RA
authors.lastname: Boykins
authors.forename: Peter S
authors.initials: PS
authors.lastname: Backlund
authors.forename: Guiyu
authors.initials: G
authors.lastname: Wang
authors.forename: Hao-Chia
authors.initials: HC
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002364:Caseins / Q000032:analysis; D003544:Cysteic Acid / Q000031:analogs & derivatives / Q000032:analysis*; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D011492:Protein Hydrolysates / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism; D017421:Sequence Analysis; D014357:Trypsin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D002364:Caseins; D007202:Indicators and Reagents; D010455:Peptides; D011492:Protein Hydrolysates; C469782:methylcysteic acid; D010769:Phosphothreonine; D010768:Phosphoserine; D003544:Cysteic Acid; D011494:Protein Kinases; D014357:Trypsin