Targeted proteomics is a technique in which target proteins are quantified using mass spectrometry, and it has several advantages over non-targeted proteomics regarding sensitivity, quantitation, and throughput. Targeted proteomics is a technology that complements non-targeted proteomics by accurately quantifying candidate proteins identified by quantitative proteomics. Furthermore, it has advantages over antibody-based protein quantification regarding high specificity and multimolecular quantification, making it effective for quantifying proteins for which no antibodies are available. Because of these characteristics, it is used to validate candidate biomarker proteins and quantify proteins with post-translational modifications. However, targeted proteomics requires knowledge that is different from that required for non-targeted proteomics. In this article, we summarize the technical basics of targeted proteomics, including its technical outline and expertise, and discuss our achievements in the application of targeted proteomics.

Hydrogen deuterium exchange-MS and Footprinting-MS techniques are capable of predicting the binding site of a binding partner mapped on the protein’s structure deposited in the protein data bank (PDB). Despite these predictions, it frequent discrepancies in the structure information obtained between these MS techniques and PDB data. In this study, we evaluated the structure information derived from MS with 27 high-resolution X-ray crystal structures of human serum albumin to discuss what might be responsible for the discrepancy and to clarify the possibility that structural information obtained by MS can be used to analyze ensemble states in solution. Our findings suggested that MS may become a useful tool for elucidating the structural states of protein conformations in solution under physiological conditions.

We independently constructed fecal proteome analysis, and investigated the effects of intestinal bacteria on host-derived proteins in the digestive tract. Through comprehensive proteomic analysis of cecal contents from germ-free (GF) mice lacking bacteria, and specific-pathogen-free mice, total of 713 mouse-derived proteins were identified, and the presence of anionic trypsin-2 (PRSS2) was notably high in the cecal contents of GF mice. Elevated levels of proteases, such as trypsin, in the distal intestine have been associated with intestinal pathological conditions. Nevertheless, the factors and mechanisms responsible for regulating proteases within the intestinal lumen have remained enigmatic. By integrating proteomics into microbiome research, we demonstrated that Paraprevotella strains, such as Paraprevotella clara (P. clara) isolated from the fecal microbiome of healthy human donors, served as potent trypsin-degrading commensals. Mechanistically, P. clara recruit trypsin to its bacterial surface through a type IX secretion system, thereby promoting trypsin autolysis. Furthermore, P. clara enhances the host’s defense mechanisms against pathogens such as bacteria and viruses by degrading trypsin that flows into the colon after protein digestion in the small intestine. The results of this research are expected to contribute to the prevention and treatment of bacterial and viral infections by trypsin-degrading bacteria. The intricacies of the gut microbiome pose a considerable challenge, given its complex ecosystem. However, proteomics will undoubtedly play a pivotal role in elucidating its mysteries in the next generation of the gut bacterial researches.