Abstract
Analysis of posttranslational modifications (PTMs) of proteins is of major interest in current biomedical research due to the multiple roles played by PTMs in protein regulation and function. In general this is a complex problem because first and foremost there are large numbers of potential PTMs in every protein that result in myriads of possible combinations of functional enzymes. In this chapter we present a general method for PTM analysis based on specifically labeling modified and unmodified samples with distinguishable fluorescent dyes followed by protein fractionation. The method is open to further refinements; in particular there are no computer programs tailored for this type of analysis, and in multiple cases little or nothing is known about a specific PTM, about how to alter such modification, or about successful labeling of the target amino acid. We present this chapter with two goals in mind, first to share with other scientists some of our experiences in this field, and second to invite those interested in the subject to bring their own contributions to an area that should be further explored and enhanced in order to create a large tool kit for PTM analysis. Although we present just four examples of this technology, in principle, any PTM can be targeted for analysis using the general principles delineated here.
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References
International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431(7011):931–945. doi:10.1038/nature03001
International HapMap C, Altshuler DM, Gibbs RA, Peltonen L, Altshuler DM, Gibbs RA, Peltonen L, Dermitzakis E, Schaffner SF, Yu F, Peltonen L, Dermitzakis E, Bonnen PE, Altshuler DM, Gibbs RA, de Bakker PI, Deloukas P, Gabriel SB, Gwilliam R, Hunt S, Inouye M, Jia X, Palotie A, Parkin M, Whittaker P, Yu F, Chang K, Hawes A, Lewis LR, Ren Y, Wheeler D, Gibbs RA, Muzny DM, Barnes C, Darvishi K, Hurles M, Korn JM, Kristiansson K, Lee C, McCarrol SA, Nemesh J, Dermitzakis E, Keinan A, Montgomery SB, Pollack S, Price AL, Soranzo N, Bonnen PE, Gibbs RA, Gonzaga-Jauregui C, Keinan A, Price AL, Yu F, Anttila V, Brodeur W, Daly MJ, Leslie S, McVean G, Moutsianas L, Nguyen H, Schaffner SF, Zhang Q, Ghori MJ, McGinnis R, McLaren W, Pollack S, Price AL, Schaffner SF, Takeuchi F, Grossman SR, Shlyakhter I, Hostetter EB, Sabeti PC, Adebamowo CA, Foster MW, Gordon DR, Licinio J, Manca MC, Marshall PA, Matsuda I, Ngare D, Wang VO, Reddy D, Rotimi CN, Royal CD, Sharp RR, Zeng C, Brooks LD, McEwen JE (2010) Integrating common and rare genetic variation in diverse human populations. Nature 467(7311):52–58. doi:10.1038/nature09298
Doniger SW, Kim HS, Swain D, Corcuera D, Williams M, Yang SP, Fay JC (2008) A catalog of neutral and deleterious polymorphism in yeast. PLoS Genet 4(8), e1000183. doi:10.1371/journal.pgen.1000183
Ayoubi TA, Van De Ven WJ (1996) Regulation of gene expression by alternative promoters. FASEB J 10(4):453–460
Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6(5):386–398. doi:10.1038/nrm1645
Walsh C (2005) Posttranslational modification of proteins: expanding nature's inventor, 1st edn. Roberts and Company Publishers, Englewood, CO
Khoury GA, Baliban RC, Floudas CA (2011) Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep 1. doi:10.1038/srep00090
Lu CT, Huang KY, Su MG, Lee TY, Bretana NA, Chang WC, Chen YJ, Chen YJ, Huang HD (2013) DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications. Nucleic Acids Res 41(Database issue):D295–D305. doi:10.1093/nar/gks1229
Shannon DA, Weerapana E (2013) Orphan PTMs: rare, yet functionally important modifications of cysteine. Biopolymers. doi:10.1002/bip.22252
Diez R, Herbstreith M, Osorio C, Alzate O (2010) 2-D fluorescence difference gel electrophoresis (DIGE) in neuroproteomics. In: Alzate O (ed) Neuroproteomics. Frontiers in Neuroscience, Boca Raton, FL
DeKroon RM, Robinette JB, Osorio C, Jeong JS, Hamlett E, Mocanu M, Alzate O (2012) Analysis of protein posttranslational modifications using DIGE-based proteomics. Methods Mol Biol 854:129–143. doi:10.1007/978-1-61779-573-2_9
Drisdel RC, Green WN (2004) Labeling and quantifying sites of protein palmitoylation. Biotechniques 36(2):276–285
DeKroon RM, Osorio C, Robinette JB, Mocanu M, Winnik WM, Alzate O (2011) Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype. J Proteome Res 10(4):1632–1644. doi:10.1021/pr1009788
Rubel CE, Schisler JC, Hamlett ED, DeKroon RM, Gautel M, Alzate O, Patterson C (2013) Diggin' on u(biquitin): a novel method for the identification of physiological E3 ubiquitin ligase substrates. Cell Biochem Biophys 67(1):127–138. doi:10.1007/s12013-013-9624-6
Ehlers MD (2003) Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat Neurosci 6(3):231–242. doi:10.1038/nn1013
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Hamlett, E.D., Osorio, C., Alzate, O. (2015). Identification and Characterization of Protein Posttranslational Modifications by Differential Fluorescent Labeling. 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_91
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DOI: https://doi.org/10.1007/7657_2015_91
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