Improvement of the MS/MS fragment ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine

Anal Chem. 2004 Oct 1;76(19):5894-902. doi: 10.1021/ac049374r.

Abstract

Tandem mass spectrometry (MS/MS) is a powerful tool for peptide sequencing and characterization. However, the selective cleavage at acidic residues, aspartic acid, and glutamic acid prevents the generation of enough product ions to elucidate the entire sequence. We attempted to solve the problem by converting the residues into the corresponding amides, asparagine and glutamine. The amidation suppressed the cleavage at the converted residues, and the product ions derived from dissociation at other sites became abundant. Incorporation of nitrogen isotope (15)N in the amine constituent for amidation minimized the mass change from -0.984 016 to +0.013 019, allowing easy discrimination of acidic and amide residues in the original sequences by MS/MS database search. In addition, the amidated and unchanged peptides had the same nominal mass, even when the transformation was incomplete, which was approximately 70% in the current condition. The unmodified acidic residues remaining were rather useful to give marker fragments by the dominant dissociation. These results demonstrate that (15)N-amidation is effective in improving the performance of MS/MS to elucidate amino acid sequences of peptides.

MeSH terms

  • Acids / chemistry*
  • Amination
  • Amines / chemistry*
  • Amino Acid Sequence
  • Animals
  • Cattle
  • Horses
  • Ions / analysis
  • Ions / chemistry
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Peptides / analysis*
  • Peptides / chemical synthesis
  • Peptides / chemistry*
  • Serum Albumin, Bovine

Substances

  • Acids
  • Amines
  • Ions
  • Nitrogen Isotopes
  • Peptides
  • Serum Albumin, Bovine