Tandem mass spectrometry of ribonuclease A and B: N-linked glycosylation site analysis of whole protein ions.
Recently, an approach for the "top down" sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [M + 12H]12+ to [M + 5H]5+ ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that "protect" the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the "protein fragment ion mass fingerprint".
Reid
Gavin E
GE
Department of Chemistry, Purdue University, West Lafayette, Indiana, 47907-1393, USA. gavinreid@purdue.edu
Stephenson
James L
JL
McLuckey
Scott A
SA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D015394:Molecular Structure; D012259:Ribonuclease, Pancreatic / Q000032:analysis* / Q000737:chemistry; D012260:Ribonucleases / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D006023:Glycoproteins; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic