Protein Structural Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry with Top-down Electron Capture Dissociation

Abstract

This study tested the feasibility of observing H/D exchange of intact protein by top-down electron capture dissociation (ECD) mass spectrometry for the investigation of protein structure. Ubiquitin is selected as a model system. Local structural information was obtained from the deuteration levels of c and z. ions generated from ECD. Our results showed that α-helix region has the lowest deuteration level and the C-terminal fraction containing a highly mobile tail has the highest deuteration level, which correlates well with previous X-Ray and HDX/NMR analyses. We studied site-specific H/D exchange kinetics by monitoring H/D exchange rate of several structural motives of ubiquitin. Two hydrogen bonded β-strands showed similar HDX rates. However, the outer β-strand always has higher deuteration level than the inner β-strand. The HDX rate of the turn structure (residues 8-11) is lower than that of β-strands (residues 1-7 and residues 12-17) it connects. Although isotopic distribution gets broader after H/D exchange which results in a limited number of backbone cleavage sites detected, our results demonstrate that this method can provide valuable detailed structural information of proteins. This approach should also be suitable for the structural investigation of other unknown proteins, protein conformational changes, as well as protein-protein interactions and dynamics.

Mass Spectrometry Methods for Studying Structure and Dynamics of Biological Macromolecules

“…It has now been recognized that collisional activation tends to induce H/D migration along the polypeptide backbone (“scrambling”), thereby randomizing the exchange pattern . In contrast, ECD and ETD allow top-down experiments to be conducted without any significant scrambling. ,, The backbone amide deuteration status can be obtained by subtractive analysis of consecutive c and z • ions, similar to the strategy outlined above for solution phase digestion data. , Often the N- and C-terminal fragment ion series overlap, providing an internal consistency check of the deuteration data obtained. The nonspecific nature of electron-based dissociation provides excellent sequence coverage.…”

“…137 In contrast, ECD and ETD allow top-down experiments to be conducted without any significant scrambling. 46,138,139 The backbone amide deuteration status can be obtained by subtractive analysis of consecutive c and z • ions, similar to the strategy outlined above for solution phase digestion data. 70,88 Often the N-and C-terminal fragment ion series overlap, providing an internal consistency check of the deuteration data obtained.…”

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

“…Improvements to the Pre-LC steps were reported and include work describing the enzymatic digestion step, − disulfilde bond reduction using electrochemical cells, , affinity capture, methodological consideration for reducing back-exchange in MALDI and electrospray, and offline fast mixing for HDX labeling . Reports of methods improvements in the LC-MS portions of the experiment included subzero cooling to minimize back exchange during LC/MS, , descriptions of top-down HDX MS studies using ECD or ETD fragmentation − where the goal is the elimination of proteolytic digestion and/or improvement to the spatial resolution of the exchange information, microfluidic systems for exchange, LC and MS, , work related to improving separation and understanding LC column parameters, − comparison of various platforms for HDX MS, and description of fine isotope structure in HDX MS . A number of software improvements was made including new software packages or updates to existing software, ,− studies of how to process data, , software approaches to improve spatial resolution, , and other work on computational and processing improvements. − Several other interesting studies, including methods descriptions, histidine exchange, and useful tools, were published. − …”

On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)

“…Indeed, Ubiquitin is a globular 1 16 comprising a mixed ␤ sheet. The ␣ helix backbone amide hydrogens are virtually nonexchangeable because of their strong interaction with adjacent carbonyl oxygens, whereas those of the ␤ strands are slow exchanging (20). These structures, together with uncorrected back exchange (21), limit the maximum deuterium incorporation observed to 53%.…”