Influence of Basic Residue Content on Fragment Ion Peak Intensities in Low-Energy Collision-Induced Dissociation Spectra of Peptides

Abstract

The primary utility of trypsin digestion in proteomics is that it cleaves proteins at predictable locations, but it is also notable for yielding peptides that terminate in basic arginine and lysine residues. Tryptic peptides fragment in ion trap tandem mass spectrometry to produce prominent Cterminal y series ions. Alternative proteolytic digests may produce peptides that do not follow these rules. In this study, we examine 2568 peptides generated through proteinase K digestion, a technique that produces a greater diversity of basic residue content in peptides. We show that the position of basic residues within peptides influences the peak intensities of b and y series ions; a basic residue near the N-terminus of a peptide can lead to prominent b series peaks rather than the intense y series peaks associated with tryptic peptides. The effects of presence and position for arginine, lysine, and histidine are explored separately and in combination. Arg shows the most dominant effects followed by His and then by Lys. Fragment ions containing basic residues produce more intense peaks than those without basic residues. Doubly charged precursor ions have generally been modeled as producing only singly charged fragment ions, but fragment ions that contain two basic residues may accept both protons during fragmentation. By characterizing the influence of basic residues on gasphase fragmentation of peptides, this research makes possible more accurate fragmentation models for peptide identification algorithms.Trypsin digestion is a standard step in many proteomic experiments. Proteins digested by this enzyme are predominantly cleaved C-terminal to arginine and lysine residues, yielding predictable collections of peptides.1 Tryptic peptides are particularly favored for tandem mass spectrometry because they yield spectra with prominent y series ions. Database search algorithms such as SEQUEST 2 have been tuned to work best with tryptic peptides. Alternative digestion techniques can be useful to analyze proteins that do not have many suitable cleavage sites for trypsin (such as membrane proteins) or to increase the diversity of peptides covering a protein region of interest. The cleavage specificities of other digestion enzymes may yield peptides that fragment differently than those produced by trypsin.One technique showing promise for proteomics is proteinase K digestion. 3 Proteinase K's specificity is more variable than trypsin, favoring amino acids with aromatic side chains as cleavage sites but targeting other residues as well. 4 The digestion time required is less than that of trypsin. Because of proteinase K's broader specificity, the diversity of peptides yielded by these digests is higher than in tryptic digests.The most common type of fragmentation used for proteomics is low-energy collision-induced dissociation (CID). In this technique, peptide ions of a particular mass-to-charge ratio are isolated and collided with noble gas atoms. Collisions add energy to the peptide ions and they fragment. The cleavage o...

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A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

“…From recent studies it is known that peptides containing internal basic residues (Arg, homo-Arg, or His) do not fragment as readily as typical tryptic fragments [25]. MALDI TOF/TOF analysis of the underivatized synthetic peptide ELAQYNVEVH-PYTVRK yielded an incomplete y-ion series, in which the y 6 -, y 7 -, y 8 -, and y 15 -ion were the most abundant fragment ions (Figure 4b).…”

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