![]() Further in vivo digest-ibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases. In vitro digestibility of the protein using SGF and SIF showed the disappearance of~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of 16% from N-terminal region. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% β-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The principles of these techniques are discussed in the chapter and examples of their applications are also provided. In the analysis of these compounds, spectroscopic methods are also being developed. Capillary gas chromatography and high performance liquid chromatography coupled with various detection systems and mass spectrometers enable identification of individual compounds. Content of tocochromanols in food may be determined using a wide range of analytical techniques. This chapter also includes an overview of the properties and occurrence of other forms of tocols (including: tocomonoenols, tocodienols, and plastochromanol-8). Tocopherols and tocotrienols are not the only compounds classified as tocol derivatives. In addition to the antioxidant role of vitamin E and protection against oxidative stress, the “non-antioxidant” functions of vitamin E, including cell signaling and antiproliferation, is also described. In food and biological systems, the main function of tocochromanols is antioxidant action – they deactivate free radicals and protect lipids from peroxidation (autoxidation). We also identify emerging technologies and methods that can be integrated into the discovery platform toward the comprehensive study of endogenous peptide hormones. The critical challenges and corresponding measures for peptidomics from the steps of sample preparation, peptide extraction, and separation to the MS data acquisition and analysis are also discussed. Although the peptidomics approach is expected to discover novel peptide hormones unbiasedly, only a limited number of successful cases have been reported. Here, we summarize and discuss the roles of targeted and untargeted MS in discovering peptide hormones using bioassay‐guided purification, bioinformatics screening, or the peptidomics‐based approach. However, it is difficult to directly identify these peptide hormones due to their diverse characteristics, dynamic regulations, low abundance, and existence in a complicated biological matrix. Mass spectrometry (MS) has been developed to be a powerful technology for identifying and quantifying peptides in a highly efficient manner. Endogenous peptide hormones represent an essential class of biomolecules, which regulate cell–cell communications in diverse physiological processes of organisms.
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