We recently developed a method to selectively derivatize single peptides at their N-termini as their corresponding tris-[2,4,6,-trimethoxyphenyl]-phosphonium acetates (TMPP-Ac). The objective of this project is to extend this procedure to peptide mixtures, specifically to proteolytic digests. We have now demonstrated that tryptic digests(containing peptides with a C-terminal arginine or lysine residue)of proteins at the 10 picomole level can be treated with the TMPP-Ac succinimide reagent to produce N-terminally TMPP-Ac N-terminally modified peptides. With pH control lysine amino group derivatization can be avoided. Direct MALDI-MS analysis of the derivatized digest allows only a subset of the peptide products to be detected. However, after microbore HPLC separation, an increased number of derivatized peptides can be identified, though complete sequence coverage in a single proteolytic digest has not yet been achieved. MALDI-PSD-MS of individual fixed-charge TMPP-Ac peptides produces a complete series of a-type ions for up to 25-mers. Current work is directed at improving derivatization coverage of proteolytic peptides in the mixture.
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