Rapid and Easy Enrichment Strategy for Naturally Acetylated N Termini Based on LysN Digestion and Amine-Reactive Resin Capture.

Abstract

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.