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Transglutaminase 2 (TG2) is a ubiquitous mammalian enzyme that is implicated in a variety of physiological processes and human diseases. Normally, extracellular TG2 is catalytically dormant due to formation of an allosteric disulphide bond between Cys370 and 371 of the enzyme. In this protocol, we describe a method to reduce this disulphide bond in living mice and to monitor the resulting in vivo TG2 activity. Briefly, exogenous thioredoxin-1 protein (TRX) is prepared and administered as a specific, physiologically relevant reductant of the Cys370-371 disulphide along with the small molecule 5-biotinamidopentylamine (5-BP) as a TG2 activity probe. Tissue cryosections are then analyzed by immunohistochemistry to ascertain the extent of 5-BP incorporation, which serves as a record of the redox state of TG2 in vivo. This protocol focuses on the modulation and measurement of TG2 in the small intestine, but we encourage investigators to evaluate it in their organ(s) of interest.

The field of peptidomics has been under development since its start more than 20 years ago. In this chapter we provide a personal outlook for future directions in this field. The applications of peptidomics technologies are spreading more and more from classical research of peptide hormones and neuropeptides towards commercial applications in plant and food-science. Many clinical applications have been developed to analyze the complexity of biofluids, which are being addressed with new instrumentation, automization, and data processing. Additionally, the newly developed field of immunopeptidomics is showing promise for cancer therapies. In conclusion, peptidomics will continue delivering important information in classical fields like neuropeptides and peptide hormones, benefiting from improvements in state-of-the-art technologies. Moreover, new directions of research such as immunopeptidomics will further complement classical omics technologies and may become routine clinical procedures. Taken together, discoveries of new substances, networks, and applications of peptides can be expected in different disciplines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The study of urinary peptidome is an important area of research, which concerns the characterization of endogenous peptides, as well as the identification of biomarkers for a wide range of socially significant diseases. First of all, this relates to renal and genitourinary pathologies and/or pathologies associated with proteinuria, such as kidney diseases, bladder, prostate and ovarian cancers, diabetic nephropathy, and pre-eclampsia. Unlike proteins, peptides do not require proteolytic hydrolysis, can be analyzed in their native form and can provide certain information about occurring (patho)physiological processes. Mass spectrometry (MS)-based approaches are the most unbiased and sensitive instruments with high multiplexing capacity and provided most of the current information about endogenous urine peptides. However, despite the large number of urine peptidomic studies, there are certain issues related to the insufficient comparability of their results due to the lack of consistent approaches to their interpretation. Also the development of a custom project-specific protein library for endogenous peptides search and identification is another important point that should be noted in the context of high-throughput peptidomic analysis. Here we propose the custom-specific urinary protein database and the grouping of endogenous urinary peptides with overlapping sequences as useful tools, which can facilitate the acquisition and analysis of LC-MS peptidomic data, as well as the comparison of results of different studies, which should facilitate their more efficient further application., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)