Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry.

Rationale: Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies have recently been published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine-reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed.

Methods: We present here a mass spectrometry (MS)-based study to evaluate and optimize the trypsin proteolysis on a guanidinated-modified protein. Trypsin proteolysis was studied using different amounts of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution.

Results: We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine-modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counterparts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS.

Conclusions: We found enzyme trypsin is active in the guanidinated form of the protein depending on the enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on homoarginine-modified proteins and our simple and versatile solid-phase capture strategy could be very useful for enrichment and sequence determination of proteins N-termini by MS.