Top-Down Analysis of In-Source HDX of Native Protein Ions

Abstract

Hydrogen/deuterium exchange (HDX) is used in protein biophysics to probe folding dynamics, intermolecular interactions, epitope and other mapping. A typical procedure often involves HDX in buffered D2O solution followed by pepsin digestion, and liquid chromatography/electrospray ionization mass spectrometry analysis. In this work, HDX of protein ions was conducted in the ESI source. Both native electrospray droplets of ubiquitin and denatured myoglobin were exposed to D2O vapor in the source region of a Bruker SolariX 12T FTICR-mass spectrometer. Electron capture dissociation was used to assess deuterium incorporation at the residue level. This in-source HDX, on the millisecond-time scale, exchanges side-chain hydrogens and fast-exchanging amides compared to conventional minutes-to-hours HDX of backbone hydrogens in solution with less sample preparation (i.e., no D2O/protein mixing and incubation, no quenching, protein digestion, or LC separation).

In-Line Dopant Generation for Atmospheric Pressure Ionization Mass Spectrometry

“…HDX through the reaction between primary and supplemental electrospray microdroplets has been shown to be faster than the reaction in the bulk solution; it may follow that the reaction between microdroplets and the gas-phase deuterated solvent is faster still if the experiment is repeated at a nanoscale. The observed exchange in native-spray conditions exposed to similar sheath gas flow rates was comparable to that of prior observations . The number of observed deuterium exchanges decreased with the native-spray ubiquitin charge state to reflect the folding of the molecule, the +7 form producing a 10 Da shift, +6 form producing a 9 Da shift, and the +5 form producing an 8 Da shift.…”

“…The observed exchange in native-spray conditions exposed to similar sheath gas flow rates was comparable to that of prior observations. 32 The number of observed deuterium exchanges decreased with the native-spray ubiquitin charge state to reflect the folding of the molecule, the +7 form producing a 10 Da shift, +6 form producing a 9 Da shift, and the +5 form producing an 8 Da shift. Both analytes in acidified solutions had deuterium exchange patterns relative to the charge state that were not primarily influenced by molecular folding.…”

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…Unlike several other biophysical techniques, HDX-MS possesses the advantages of no or very high size limit [ 8 ] and is useful for studying individual proteins as well as large complexes [ 34 , 35 ]. This is mainly due to the application of proteolysis [ 14 ] and radical-induced fragmentation (electron transfer and electron capture dissociation respectively; ETD and ECD) in the course of analysis [ 36 , 37 , 38 ]. HDX-MS is en route to becoming a routine analytical technique in life science research and drug discovery [ 26 ].…”

Multiplexed Conformationally-Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation

“…The protein ubiquitin has previously been used as model for in source and gas-phase HDX. 39,[46][47] As such, we began our characterization of in-trap HDX with on-the-fly ECD fragmentation by examining various charge states of ubiquitin produced by nESI from aqueous ammonium acetate and water/methanol/acid solutions. For example, isolation of the 6 + charge state by the quadrupole with and without the addition of ND3 in the trap cell is shown in Figure 2.…”