In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation

Analysis
of protein C termini is very important for functional
annotations of proteomes, while proteome-wide C termini analysis still
poses substantial challenges. Here we described a simple and robust
strategy for specific isolation of protein C termini based on LysC
digestion and site-selective dimethylation to deplete N-terminal and
internal peptides by scavenger materials. The performance of LysC
digestion and conditions of site-selective dimethylation and resin
coupling were discussed in detail. Then the strategy was successfully
applied to the characterization of protein C termini of HeLa cells.
A total of 781 protein C termini were identified with a 300 μg
digest in our study, among which 38.9% were actually not identifiable
using current trypsin digestion-based methods due to their inappropriate
peptide length for MS analysis, indicating that our method was highly
complementary to the existing methods. The enrichment procedure was
rapid and easy to operate and could afford a very good identification
efficiency by obtaining the largest C termini data set of the human
proteome with the least sample loading. This method was without bias
toward physicochemical properties of peptides. Moreover, a peptide-centric
database was first introduced to analyze protein C termini, which
effectively improved the accuracy and speed of the database search.
Therefore, our method can be used to effectively and selectively isolate
protein C termini and contributes to the global annotation of C terminomes.