Tandem Mass Spectrometry of Ribonuclease A and B: N-linked Glycosylation Site Analysis of Whole Protein Ions

Anal Chem. 2002 Feb 1;74(3):577-83. doi: 10.1021/ac015618l.

Abstract

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".

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • Glycoproteins / analysis
  • Glycoproteins / chemistry
  • Glycosylation
  • Molecular Structure
  • Ribonuclease, Pancreatic / analysis*
  • Ribonuclease, Pancreatic / chemistry
  • Ribonucleases / analysis*
  • Ribonucleases / chemistry
  • Spectrometry, Mass, Electrospray Ionization / instrumentation
  • Spectrometry, Mass, Electrospray Ionization / methods*

Substances

  • Glycoproteins
  • Ribonucleases
  • ribonuclease B
  • Ribonuclease, Pancreatic