Real-time HD Exchange Kinetics of Proteins from Buffered Aqueous Solution with Electrothermal Supercharging and Top-Down Tandem Mass Spectrometry
Autor: | Evan R. Williams, Catherine C. Going, Zijie Xia |
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Rok vydání: | 2016 |
Předmět: |
H/D exchange
Spectrometry Mass Electrospray Ionization Protein Conformation Electrospray ionization Analytical chemistry 010402 general chemistry Tandem mass spectrometry 01 natural sciences Article Analytical Chemistry Ion Medicinal and Biomolecular Chemistry Reaction rate constant Tandem Mass Spectrometry Structural Biology Spectroscopy Chromatography Aqueous solution Spectrometry Chemistry Electrospray Ionization 010401 analytical chemistry Supercharging Proteins Deuterium Exchange Measurement Mass Random coil 0104 chemical sciences Electron-transfer dissociation Kinetics Electrothermal supercharging Reagent Physical Chemistry (incl. Structural) |
Zdroj: | Journal of the American Society for Mass Spectrometry, vol 27, iss 6 |
ISSN: | 1044-0305 |
DOI: | 10.1007/s13361-016-1350-z |
Popis: | Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX. Rate constants for HDX of ubiquitin are obtained with a spatial resolution of ~1.3 residues with ETS and electron transfer dissociation of the 10+ charge-state using a single capillary containing a few μL of protein solution in which HDX continuously occurs. HDX protection at individual residues with ETS HDX is similar to that with reagent supercharging HDX and with solution-phase NMR, indicating that the high spray potentials required to induce ETS do not lead to HD scrambling. Graphical Abstract ᅟ. |
Databáze: | OpenAIRE |
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