Abstract
Posttranslational modifications (PTMs) of proteins are important determinants of their biological functions. Proteins undergo various PTMs throughout their life span. Some of these modifications are of a temporary nature and may control rapid on/off rates for activation or inactivation of particular proteins. Other types of PTMs are of a permanent nature. Those attachments take place upon protein synthesis and may substantially alter protein structures and function, and are removed only upon protein hydrolysis. In these modifications, the moieties of the modifier molecule are most likely bound covalently. Here we discuss such a type of PTM with a polyester, poly-(R)-3-hydroxybutyrate (PHB). PHB is a ubiquitous homopolymer that is present in all living organisms. In animals PHB was specifically found in the liver, kidney, heart, and brain. However, what role PHB plays in these tissues is not well understood. As a polymer-electrolyte, PHB has been recognized in mediating ion transport across the membrane, and thus it may be implicated in various signaling pathways carried in the central and peripheral nervous systems. In this chapter, we present a protocol for determination of PHB modification of the mammalian ion channel, TRPM8. The TRPM8 channel is the cold and menthol receptor in the peripheral nervous system, and an important mediator of pain stimuli. The procedures to determine the PHB moieties on the specific amino acids of TRPM8, including protein isolation, purification, digestion, mass spectrometry analysis, and database search, will be outlined here. Herein we also discuss the challenges to resolving PHBylated peptides that may arise due to the fragile chemical structure of the polyester and its disintegration during the experimental procedures and mass spectrometry.
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Acknowledgements
This work was supported by the American Heart Association SDG-2640223 grant to E. Z., and the National Institutes of Health grant R01GM098052 to E. Z. The project described was supported by a grant from the Foundation of UMDNJ and a grant (P30NS046593) from the National Institute of Neurological Disorders and Stroke. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. The authors report no conflict of interest.
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Liu, T., Chen, W., Pan, S., Cui, C., Li, H., Zakharian, E. (2015). Determination of Polyhydroxybutyrate (PHB) Posttranslational Modifications of Proteins Using Mass Spectrometry. In: Grant, J., Li, H. (eds) Analysis of Post-Translational Modifications and Proteolysis in Neuroscience. Neuromethods, vol 114. Humana Press, New York, NY. https://doi.org/10.1007/7657_2015_86
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DOI: https://doi.org/10.1007/7657_2015_86
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