Jeremiah Tipton - Sequential Proteolysis and High-Field FTICR MS To Determine Disulfide Connectivity and 4-Maleimide TEMPO Spin-Label Location in L126C GM2 Activator Protein

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      Publication Details (including relevant citation   information):

      Tipton, J.D.; Carter, J.D.; Mathias, J.D.;   Emmett, M.R.; Fanucci, G.E. and Marshall, A.G., Analytical   Chemistry, 81 (18), 7611-7617 (2009)


      The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic   accessory protein involved in the degradation of neuronal   gangliosides. Genetic mutations of GM2AP can disrupt ganglioside   catabolism and lead to deadly lysosomal storage disorders.   Crystallography of wild-type GM2AP reveals 4 disulfide bonds and   multiple conformations of a flexible loop region that is thought   to be involved in lipid binding. To extend the crystallography   results, a cysteine construct (L126C) was expressed and modified   with 4-maleimide TEMPO for electron paramagnetic resonance (EPR)   studies. However, because a ninth cysteine has been added by   site-directed mutagenesis and the protein was expressed in E.   coli in the form of inclusion bodies, the protein could   misfold during expression. To verify correct protein folding and   labeling, a sequential multiple-protease digestion, nano-liquid   chromatograph (LC) electrospray ionization 14.5 T Fourier   transform ion cyclotron resonance mass spectrometry assay was   developed. High-magnetic field and robust automatic gain control   results in subppm mass accuracy for location of the spin-labeled   cysteine and verification of proper connectivity of the four   disulfide bonds. The sequential multiple protease digestion   strategy and ultrahigh mass accuracy provided by FTICR MS allow   for rapid and unequivocal assignment of relevant peptides and   provide a simple pipeline for analyzing other GM2AP constructs.

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