Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins.
The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.
Hauser
Nicolas J
NJ
Department of Chemistry, University of Wyoming, Laramie, WY 82071, USA.
Han
Hongling
H
McLuckey
Scott A
SA
Basile
Franco
F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004583:Electrons; D013058:Mass Spectrometry; D008872:Microwaves; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization
D010446:Peptide Fragments; D010455:Peptides