Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing.
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.
Li
Wei
W
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.
Boykins
Robert A
RA
Backlund
Peter S
PS
Wang
Guiyu
G
Chen
Hao-Chia
HC
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
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