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.
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http://dx.doi.org/10.3724/SP.J.1123.2021.03030 | DOI Listing |
Front Immunol
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Jiangsu Engineering Research Center of Biological Data Mining and Healthcare Transformation, Xuzhou Medical University, Xuzhou, China.
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January 2025
Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi, U.P. 221005 India.
Unlabelled: Potato peels are one of the most under-utilized wastes which can be highly beneficial to mankind. The red potato peel powder was prepared by using tray drying and vacuum-oven drying method. The proximate analysis of red potato peel powder was conducted followed by its characterization which includes FT-IR, XRD, TGA, DSC, and SEM.
View Article and Find Full Text PDFJ Food Sci Technol
January 2025
Food Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow, Uttar Pradesh 226001 India.
Unlabelled: Solid phase extraction technique is a widely used sample preparation technique for the extraction of components from complex food matrices. However, there are several parameters in SPE that leads to low recovery, and reproducibility, insufficiently clean extracts and evaporation of volatile compounds. These drawbacks can be addressed through the use of innovative techniques and instrumentation that offers improved efficiency and accuracy for isolation of active constituents from food and beverage samples.
View Article and Find Full Text PDFInt J Clin Pediatr Dent
December 2024
Department of Pedodontics and Preventive Dentistry, Vivekanandha Dental College for Women, Namakkal, Tamil Nadu, India.
Background And Objective: During the last 200 years, there have been many changes in the way of performing endodontic treatment. The increased demand from patients for saving their teeth has led to the development of various innovative equipment and advances in the field of material sciences. Thus, the standard protocol of endodontic treatment has undergone several modifications.
View Article and Find Full Text PDFInt J Dent
January 2025
Department of Orthodontics, School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran.
This research aimed to assess the shear bond strength (SBS) of metal brackets bonded to composite veneers using different surface preparations. One-hundred composite disks were divided into 10 different groups whereby each group combines a surface preparation (roughening or no roughening), etching agent (37% phosphoric or 9.5% hydrofluoric acid), adhesive protocol (self-etch or total-etch), and bonding agent (with or without G-Premio Bond).
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