Enrichment of N-Terminally Acetylated Peptides
N-terminally acetylated peptides can be separated from internal tryptic peptides and unmodified N-terminal peptides due to differences in charge, resulting from blockage of the N-terminal amine. SCX at low pH (<3) can resolve these species based on charge state, but it is not completely selective since peptides of similar or lower positive charge such as phosphopeptides, C-terminal peptides, or Glu/Asp-containing peptides elute in a similar retention window. This can be improved by the use of LysN, which cleaves before lysine residues, to produce uncharged N-terminally acetylated peptides and singly charged internal peptides .
Step change for this strategy was achieved by introduction of a dimethylation step posttryptic digest to block free (N-terminal, lysine-e) amines thus increasing the difference in basicity between N-acetylated and the rest of peptides in the sample. One-step purification of N-acetylated peptides is achieved by solid-phase extraction, using SCX in batch mode. A key benefit of this approach is that it can be utilized with stable isotope-labeled dimethylation reagents for relative quantification, an approach which has found application to distinguish protein isoforms that are N-terminally acetylated but differ in N-terminal amino acid sequence, for example, p-actin/y-actin isoforms .