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

Abstract

Rationale Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies recently 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-based study to evaluate and optimize the trypsin proteolysis on a guanidinated modified protein. Trypsin proteolysis was studied using different amount 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 counter parts 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. Conclusion We found enzyme trypsin is active in guanidinated form of protein depending on 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 mass spectrometry.

Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8

“…The primary amines, including the amino-terminal end of the proteolytic fragments, were acetylated in the gel slice using 15% acetic anhydride (Sigma, #320102) for five hours at room temperature within the individual excised gel fragments [ 61 , 62 ]. Acetylation was stopped by adding 1 M NH 4 HCO 3 (Sigma, #40867) solution [ 61 , 62 ]. After 20 min, the gel pieces were shrunk by 100% ACN.…”

“…The eluted peptides were resuspended in 0.1% FA for sequencing by nanoLC-ESI-MS/MS using a Thermo Scientific LTQ Velos Pro ion trap mass spectrometer. R2 peptides were identified using Thermo Proteome Discoverer software (version 2.0); a database of R2Bm protein fragments was created, and a peptide was assigned as either N-terminal end or internal peptide based on the position of acetyl groups in the peptide sequence [ 61 ]. The internal peptides generated after trypsin (second) digestion will lack an acetyl group at the N-terminal end, as acetylation is performed prior to the second protease digestion step.…”

An optimized guanidination method for large‐scale proteomic studies

“…As the initial test, we carried out the reaction with BSA in gel in order to avoid the desalting procedures after guanidination. Since the homo‐Arg derivative is less suited for trypsin cleavage , the guanidinated BSA was digested using AspN instead of trypsin for precise estimation of the reaction yield. The digests of BSA and guanidinated BSA were analyzed by MALDI‐TOF MS and the results are shown in Fig.…”

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

“…It might be inferred that the digestion of the amino‐blocked protein was influenced. This was an intriguing issue as only few reports studying the digestion have been published . Accordingly, more techniques which could improve the digestion should be tried, including laser‐assisted proteolysis.…”