Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry

Abstract

Specific restricted proteolysis combined with subzero temperature HPLC and online ETD facilitates structural characterization of antibodies at high resolution.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass‐spectrometry‐based approach

“…Arguably less well‐recognized is the rapidly developing capability of MS instruments to analyze large, intact biomolecules under native‐like conditions . This, combined with coupled techniques like ion mobility spectrometry (IMS) and hydrogen/deuterium exchange (HDX) allows MS to support higher order structure analysis, in some cases at single residue resolution …”

“…[20][21][22][23][24][25] This, combined with coupled techniques like ion mobility spectrometry (IMS) 26,27 and hydrogen/deuterium exchange (HDX) [28][29][30] allows MS to support higher order structure analysis, in some cases at single residue resolution. 31,32 IMS is a relatively recent addition to the MS-based analysis toolbox, with a number of commercial instruments now including IMS technologies. IMS is essentially a gas-phase analog of electrophoresis, allowing for (usually pre-mass spectrometric) separation of analytes on the basis of size, shape, and charge.…”

Comparative higher-order structure analysis of antibody biosimilars using combined bottom-up and top-down hydrogen-deuterium exchange mass spectrometry

“…ETD/ECD based top-down HDX-MS approaches, however, are meeting this challenge, and continued improvements are expected for MS. As top-down HDX-MS continues to mature, the residue level information -as well as the intact subunit level information -will undoubtedly continue to increase. However, because of size limitations on the top-down approach, the top-down and bottom-up techniques are still likely to remain complementary [20,50,51].…”

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

“…When ionized in a mass spectrometer different protein conformations take on charge differently due to their size and shape, thus allowing one to isolate and analyze target conformers and their peptide fragments. [47][48][49] ECD also enables fragmentation of a peptide without scrambling of the deuterium label, as is the case with other fragmentation methods, meaning deuterium uptake can be analyzed with single amino acid resolution. 1,47,48 Pan et al were able to take advantage of different conformers' preferences for different charge states and select for specific isotopically labeled Aβ42 conformers using precursor ion selection enabling analysis of the Aβ42 oligomer's HDX profile with single amino acid resolution using ECD.…”