Optimization And Application Of Labeling Of Protein C-terminus By Carboxypeptidase Y

Objective : Proteolytic cleavage is a post-translational modification, which plays important roles in many biological processes, such as apoptosis and tumor cell metastasis.The identification of the cleavage events can improve our understanding of their biological functions in these processes. In recent years, although many N-terminomics strategies have been developed, there are only a few C-terminomics strategies that are used for the labeling and isolation of protein C-termini. In this study, we optimized the protein C-terminus by enzymatic labeling(Pro C-TEL) technique by increasing the efficiency of carboxypeptidase Y(CPY) labeling and affinity purification of the labeled C-terminal peptides. We further applied this approach to a complex protein mixture from E. coli to positively isolate protein C-terminal peptides and identified novel proteolytic cleavages.Then, we applied this method to study the proteolytic cleavages during the apoptosis of multiple myeloma cells. At last, in order to further improve the labeling efficiency, we explored the possibility of using CPY mutants in the labeling of protein C-terminus.Methods: Profiling of protein C-terminus by enzymatic labeling(Pro C-TEL) is a first positive labeling and isolating technique for the enrichment of protein C-terminus. In our study, through detecting the nature of protein C-terminal modification by carboxypeptidase Y at various p Hs and exploring the effect of SDS concentration on the overall efficiency of CPY-catalyzed biotinylation, we optimized the Pro C-TEL technique and applied it to a complex protein mixture from E. coli for the identification of proteolytic cleavages by LC-MS/MS. Through trypan blue staining, CCK-8 measurement and Western blotting analysis, we examined the apoptosis of multiple myeloma cells induced by5,6-dichlorobenzimidazole riboside(DRB) and identified the proteolytic cleavages occurring in this process. We further explored the effect of CPY mutation on the efficiency of CPY-catalyzed protein C-terminal labeling.Results: 1) Using a model peptide and MALDI-TOF-MS, we showed that the CPY-catalyzed C-terminal labeling was almost uniform at p H=11.5. 2) The efficiency ofthe labeling experiment is significantly increased by increasing the SDS concentration to an optimal condition in the reaction buffer and in the resolubilization buffer after methyl esterification. In the subsequent experiments, we used buffers containing 1% SDS to dissolve the esterified proteins, and kept the final SDS concentration in the labeling experiment at 0.5%. 3) The MALDI-TOF-MS and LC-MS/MS results showed that the modified procedure can be applied to isolate the C-terminal peptides from a model protein,horse myoglobin. 4) We applied this approach to a complex protein mixture from E. coli and identified more than 120 protein C-terminal peptides. 5) We applied the optimized Pro C-TEL and proteomics to identify the proteolytic cleavages in the apoptosis of multiple myeloma cells induced by DRB. From this experiment, we identified 81 and 88 biotinylated peptides from the DMSO treated sample and DRB treated sample, respectively.6) In the exploratory experiment, although we have successfully constructed and expressed the wild type and mutant CPY, the labeling efficiency was not increased by applying them in the labeling experiment.Conclusions: In our study, we have optimized the experimental procedure for protein C-terminal labeling by carboxypeptidase Y for affinity purification and MS identification of protein C-terminal peptides. We applied this approach to a complex protein mixture from E.coli and identified more than 120 protein C-terminal peptides. Thirty one of them were derived from protein internal cleavages, which might play important roles in regulating protein synthesis and redox-coupled reaction deduced from a bioinformatic analysis. Due to the extreme length of the resulting peptides from the labeling of neo-N-terminal peptides after cleavage, ~48% proteolytic cleavages detected here could not be identified if N-terminomics approaches were used. Then, the improved Pro C-TEL identified eleven new caspase cleavages during the apoptosis of a multiple myeloma cell line.This work provides a potential approach complementary to the N-terminomics for the identification of proteolytic cleavages in complex biological signaling pathways, such as apoptosis, tumor cell migration and invasion.