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J.W. Clack, F. Bai, N.M. Pedrick, F.A. Witzmann; Biochemical and Proteomic Analysis of Transducin Beta Subunit Structural Heterogeneity . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1697.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the nature of the structural heterogeneity of the beta subunit of Transducin (Tß). Methods: Partially purified Transducin was resolved using 2–D gel electrophoresis using both IPG/small–format gels and IEF tubes/large–format gels. Peptide mass fingerprinting of several different spots believed to correspond to Tß was performed. Spots were excised and proteolyzed using modified trypsin. Matrix–assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry were performed on the peptide mixture resulting from each spot. The effect of in vitro thiophosphorylation on 2–D protein pattern was investigated. Results: As many as six spots with different pIs, ranging from 5.2 to 6.1, were observed when separated using 2–D electrophoresis. MALDI Peptide mass fingerprinting determined with high probability (9–18 peptide mass matches per spot, coverage of 29–40%) that all of the spots were the same gene product, Guanine nucleotide–binding protein GI/GS/GT beta subunit 1 (P04901; Tß1). This suggested that post–translational modification is responsible for the differences in pI. Tß1 was thiophosphorylated using ATP–γ35S, resulting in a large acidic shift in a subset of Tß1. MS/MS fragmentation studies showed that an oligopeptide corresponding to amino acids 2–15 had a mass 42 daltons greater than predicted; interpretation of the product ion spectra from collisionally induced dissociation of the peptide determined that S2 was acetylated. A second doubly charged peptide corresponding to amino acids 198–209 had a mass of 1394.2 daltons, 224 daltons greater than predicted. MS/MS fragmentation data from this peptide determined that C204 is modified by an as yet undetermined modification on some variants of Tß1. Conclusions: Tß1 displays structural heterogeneity that is probably due to post–translational modifications. Preliminary experiments suggest that a subset of Tß1 variants may be thiophosphorylated, consistent with the reports of many investigators. It is probable that the N–terminal methionine of all variants of Tß1 has been cleaved from the protein and that the resulting N–terminal serine is acetylated. There also evidence for a 224 dalton modification of C204 by an unknown moiety.
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