基于精氨酸酶切的蛋白质C端肽段富集方法的优化及评估北大核心CSCD

基于聚合物的蛋白质C端反向富集策略是用于研究蛋白质C端最为广泛的策略之一。目前,基于胰蛋白酶(trypsin)切割精氨酸残基C端(ArgC型酶切)的蛋白C端组学方法对蛋白质C端的鉴定深度仍有待提高。为解决这一问题,该研究对此方法进行了优化和评估:建立了基于“V型”过滤装置的“一锅法”富集流程,避免了副反应的干扰,缩短了样本的制备时间;优化了蛋白水平乙酰化反应条件,最大限度地降低了丝氨酸、苏氨酸、酪氨酸残基上的副反应,提高了肽段鉴定的可信性;优化了基于固相萃取枪头膜片过滤柱(StageTip柱)的样品分离过程,使C端肽段的鉴定深度增加至原来的4倍。通过以上优化,按照肽段水平错误发现率(FDR)<0.01、离子分数(ion score)≥20,且C端带有乙醇胺修饰的数据筛选标准,从人HEK 293T细胞中共鉴定出696个蛋白质C端。若仅要求肽段水平FDR<0.01,鉴定数目进一步增加到933个,这是基于聚合物富集策略的蛋白质C端组学方法所得的最大数据集之一。探索了胰蛋白酶镜像酶(LysargiNase)切割精氨酸残基N端(ArgN型酶切)与不同肽段N端衍生化修饰组合对蛋白质C端鉴定数目和种类的影响,“LysargiNase酶切+肽段N端乙酰化”新策略在原有“胰蛋白酶酶切+肽段N端二甲基化”策略的基础上将鉴定蛋白质C端的种类提升了47%。综上,该研究通过对基于Arg型酶切的蛋白C端组学方法的优化,提升了C端肽段的鉴定深度,扩大了C端肽段鉴定的覆盖范围。该方法将有望成为系统性表征蛋白质C端的有力工具。

Optimization and evaluation of protein C-terminal peptide enrichment strategy based on arginine cleavage

As unique biomarkers, protein C-termini are involved in various biological processes such as protein trafficking, subcellular relocation, and signal transduction. Dysregulation of protein C-terminal status is critical during the development of various diseases, including cardiovascular, neurodegenerative, and metabolic diseases and cancer. Thus, global profiling of protein C-termini is of great value in providing mechanistic insight into biological or pathological processes, as well as for identifying potential new targets for therapeutic treatment. Polymer-based negative enrichment is a prominent C-terminomics strategy with advantages of universal applicability and parallel sample preparation. Compared with other methods of such a strategy, the profiling depth of the approaches based on enzymatic cleavage of Arg residues still needs to be improved. This greatly limits our understanding of the physiological functions and molecular mechanisms of C-termini. To add a more powerful tool for C-terminomics, Arg cleavage-based negative enrichment C-terminomics was optimized and evaluated. First, the sample preparation process was optimized. A one-pot enrichment platform based on a V-shaped filter was established, which reduced sample loss, avoided cross-contamination between reactions, and shortened sample preparation time. In addition, the protein-level acetylation conditions were investigated with the optimal labeling conditions as follows: triple coupling using 5 mmol/L Ac-NHS at pH 7.0 and 500 mmol/L ammonium for 15 min provided minimized acetylation rates(acetylation labeling efficiencies of Ser, Thr, and Tyr were lower than 4%, 2%, and 1%, respectively), along with the highest peptide-spectrum match number and satisfactory Lys labeling efficiency(up to 98%). These optimized conditions would not only minimize acetylation, but also facilitate the identification of C-terminal peptides. Second, it was speculated that the unexpected low identification rate was primarily caused by the interference of the large number of organic compounds accumulated during the peptide-level reactions, including reagents, organic buffering agents, and their complex side-reaction products. Therefore, the conditions for StageTip-based fractionation, including pH, the amount of Empore C18 beads, and the number of fractions, were optimized. As a result, by separating the sample enriched from 300 μg proteome into seven fractions, sample complexity was largely decreased and a total of 696 C-termini were identified in duplicates from strict data filtration, that is, percolator false discovery rate(FDR)<0.01, ion score≥20, and C-terminal amidation by ethanolamine. If only peptide FDR<0.01 was considered, the identified C-termini further increased to 933, which was among the largest C-terminome datasets obtained from the polymer-based strategy. Furthermore, compared with the results of a previous study, the optimized method would be a practical strategy for broader C-terminome coverage. Finally, to further broaden the coverage of the sub-C-terminome generated by Arg-specific cleavage, this study explored a new method in which ArgN-specific cleavage(cleavage at the N-terminal of Arg by LysargiNase) was combined with different N-terminal protections(dimethylation and acetylation). Among all the combinations, the additional use of the “LysargiNase+N-terminal acetylation” method increased 47% more identifications of unique C-termini on the basis of “trypsin+N-terminal demethylation” and the two covered 87% of the total C-termini. Therefore, the parallel use of the two methods would further expand the coverage of Arg-cleaved C-terminal peptides. With the analysis of the physicochemical properties of the peptides identified by the two methods, the reason why the C-terminal peptides identified by different strategies are complementary was explained. In conclusion, in this study, the optimized C-terminomics platform can deeply profile Arg cleavage-generated C-terminal peptides using a polymer-based approach. This method provides a powerful tool for the global characterization of protein C-termini.