Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies

Nowadays, bottom-up approaches are predominantly adopted in proteomics studies, which necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. A lot of efforts have been made to develop a superior digestion approach but hardly succeed, especially in large-scale proteomics studies. Herein, we report a new tandem digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been proven to be more specific and efficient than trypsin digestion. Reanalysis of our previous data (<i>Anal. Chem.</i> <b>2018</b>, <i>90</i> (3), 1554–1559) revealed that both Lys-C and Arg-C are trypsin-like proteases and perform better when considered as trypsin. In particular, for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency, and high specificity of Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically evaluated Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion specificity, efficiency, and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that the Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD009797

Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies

Nowadays, bottom-up approaches are predominantly adopted in proteomics studies, which necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. A lot of efforts have been made to develop a superior digestion approach but hardly succeed, especially in large-scale proteomics studies. Herein, we report a new tandem digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been proven to be more specific and efficient than trypsin digestion. Reanalysis of our previous data (<i>Anal. Chem.</i> <b>2018</b>, <i>90</i> (3), 1554–1559) revealed that both Lys-C and Arg-C are trypsin-like proteases and perform better when considered as trypsin. In particular, for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency, and high specificity of Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically evaluated Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion specificity, efficiency, and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that the Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD009797

Enzyme and Chemical Assisted N‑Terminal Blocked Peptides Analysis, ENCHANT, as a Selective Proteomics Approach Complementary to Conventional Shotgun Approach

Shotgun (bottom-up) approach has been widely applied in large-scale proteomics studies. The inherent shortages of shotgun approach lie in that the generated peptides often overwhelm the analytical capacity of current LC–MS/MS systems and that high-abundance proteins often hamper the identification of low-abundance proteins when analyzing complex samples. To reduce the sample complexity and relieve the problems caused by abundant proteins, herein we introduce a modified selective proteomics approach, termed ENCHANT, for enzyme and chemical assisted N-terminal blocked peptides analysis. Modified from our previous Nα-acetylome approach, ENCHANT aims to analyze three kinds of peptides, acetylated protein N-termini, N-terminal glutamine and N-terminal cysteine containing peptides. Application of ENCHANT to HeLa cells allowed to identify 3375 proteins, 19.6% more than that by conventional shotgun approach. More importantly, ENCHANT demonstrated an excellent complementarity to conventional shotgun approach with the overlap of 34.5%. In terms of quantification using data independent acquisition (DIA) technology, ENCHANT quantified 23.9% more proteins than conventional shotgun approach with the overlap of 27.6%. Therefore, our results strongly suggest that ENCHANT is a promising selective proteomics approach, which is complementary to conventional shotgun approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD007863

Systematic Optimization of C‑Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome

It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an <i>Escherichia coli</i> sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in <i>E. coli</i> in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409