title: Increasing Fragmentation of Disulfide-Bonded Proteins for Top-Down Mass Spectrometry by Supercharging.
abstract: The disulfide bond is an important post-translational modification to form and maintain the native structure and biological functions of proteins. Characterization of disulfide bond linkages is therefore of essential interest in the structural elucidation of proteins. Top-down mass spectrometry (MS) of disulfide-bonded proteins has been hindered by relatively low sequence coverage due to disulfide cross-linking. In this study, we employed top-down ESI-MS with Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD) to study the fragmentation of supercharged proteins with multiple intramolecular disulfide bonds. With charge enhancement upon the addition of sulfolane to the analyte solution, improved protein fragmentation and disulfide bond cleavage efficiency was observed for proteins including bovine β-lactoglobulin, soybean trypsin inhibitor, human proinsulin, and chicken lysozyme. Both the number and relative abundances of product ions representing disulfide cleavage increase with increasing charge states for the proteins studied. Our studies suggest supercharging ESI-MS is a promising tool to aid in the top-down MS analysis of disulfide-bonded proteins, providing potentially useful information for the determination of disulfide bond linkages.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, 90095, United States.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine at UCLA, University of California-Los Angeles, Los Angeles, California, 90095, United States ; UCLA/DOE Institute for Genomics and Proteomics, University of California-Los Angeles, Los Angeles, California, 90095, United States.
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, 90095, United States ; Department of Biological Chemistry, David Geffen School of Medicine at UCLA, University of California-Los Angeles, Los Angeles, California, 90095, United States ; UCLA/DOE Institute for Genomics and Proteomics, University of California-Los Angeles, Los Angeles, California, 90095, United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
keywords: ESI-MS; Fourier-transform ion cyclotron resonance MS; Supercharging; disulfide bond; protein; top-down MS