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

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.