Laser-assisted proteolysis for accelerating and enhancing protein N-termini analysis.

ABSTRACT

RATIONALE Targeted analysis of protein N-termini contributes to elucidating the starting sites and post-translational modifications of mature protein N-termini. Tryptic digestion is important in protein N-termini analysis, as well as in conventional bottom-up proteomics strategies. It is essential to explore a new proteolysis method for the enhancement of protein N-termini analysis. METHODS: Laser-assisted proteolysis was compared with conventional overnight proteolysis. Four standard proteins were studied as models and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 100 pg of synthesized peptide was used as internal standard for comparison of N-terminus intensity. Laser-assisted proteolysis was demonstrated to accelerate and enhance N-termini analysis. A complex mouse liver proteome sample was used to validate the effect of laser-assisted proteolysis. RESULTS: According to online database search, the number of matched peptides of four model proteins and the sequence coverage were comparable between the two proteolysis methods. Laser exposure time (40 s) could enhance the release of the N-terminus in model proteins. The number of identified N-termini in mouse liver was improved by 28.3% in the laser-assisted digest, compared to the conventional overnight digest. The time cost for digestion was shortened from overnight to 40 s. CONCLUSIONS: Laser-assisted proteolysis was demonstrated to accelerate proteolysis and enhance N-termini analysis. If laser-assisted proteolysis was integrated into protein N-termini targeted methods, the performance of those methods should be improved. Copyright © 2016 John Wiley & Sons, Ltd.