A rapid and efficient method for N-termini analysis in short-lived proteins

Abstract

A rapid and efficient method to isolate global N-termini is presented. Utilizing laser-assisted proteolysis and Fe3O4 microsphere, protein N-termini could be isolated in 4 h. The amino-blocked protein was digested by trypsin assisted by laser radiation, shortening the digest time from overnight to 40 s. Non-N-terminal peptides were characterized by a tryptic free amino in their N-term, which could be derived with sulfhydryl by traut’ s reagent efficiently and then coupled with Fe3O4 microspheres nearly completely in less than 4 h. The rapid method was beneficial for the identification of unstable N-termini in short-lived proteins. Human serum albumin was studied as a model. The N-terminus was successfully isolated from the digest within 4 h. Also, 2011 N-terminal peptides out of 936 proteins in mouse liver proteome sample were identified using liquid chromatography-tandem mass spectrometer (LC-MS/MS). This method was demonstrated as a facile and efficient N-termini enrichment method for targeted protein N-termini analysis, especially those with short half-life.