Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion

Abstract

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.

Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

“…A possible explanation for this phenomenon is that TiO 2 used for enrichment not only enriches for phosphorylated residues, but also acidic residues, leading to an increased likelihood of enriching peptides that are phosphorylated and also contain more acidic residues. These phosphopeptides are thus more likely to have missed cleavage sites because of the increased likelihood of aspartic and glutamic acid residues occurring adjacent to a tryptic cleavage sites (14). We conclude that these missed cleavages are due to phosphorylation occurring in close proximity to sites of proteolytic cleavage and recommend that a higher number of missed cleavages events should be allowed during the database searching step of phosphoproteomics experiments.…”

“…One possible explanation is that the proximity of phosphorylated residues to arginine/lysine residues (12,13) or acidic residues such as aspartic and glutamic acid (14) can hamper the efficiency of digestion by trypsin. An analysis of our data showed that, for 42% of the tryptic phosphopeptides, a phosphorylated residue was 1 or 2 aa away from the cleavage site, suggesting that the phosphorylated residue might have hindered cleavage by trypsin as noted previously (12,13).…”

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…Although the characterization of the N-terminal status of the protein and especially NAA is the main goal of this study, the approach used here has been previously used for phosphopeptide enrichment and as mentioned earlier for the characterization of C terminus peptides and N terminus pyroglutamylated peptides (61). In depth investigation of the remaining unmatched spectra uncovered other co-and posttranslational modifications or numerous artifacts such as sodium and potassium peptide cationization (62).…”

Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H+-ATPase Leads to Glucose-dependent Activation

“…Both CS and GM samples also contained peptide 896 -916 (2437.15 Da), in which Lys-C cleavage had occurred at Lys-916. The signal for 896 -916 was much less intense than that for 896 -918, presumably reflecting the fact that cleavage at Lys-916 was strongly inhibited by the flanking Glu residues (20). Furthermore, peptide 896 -916 is found in Pma2, a poorly expressed isoform of yeast plasma membrane H ϩ -ATPase (21), whereas peptide 896 -918 is unique to Pma1.…”