Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain.
Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.
Hogan
Jason M
JM
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, USA.
Pitteri
Sharon J
SJ
McLuckey
Scott A
SA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D038202:alpha-Crystallin A Chain / Q000378:metabolism*
D010455:Peptides; D011506:Proteins; D038202:alpha-Crystallin A Chain