Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.