Investigating the Influence of Glycosylation on Protein Conformation and Dynamics - Pharmaceutical Technology

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Investigating the Influence of Glycosylation on Protein Conformation and Dynamics
The authors review and discuss the influence of glycans on the conformation of a representative IgG1 biopharmceutical using H/DX-MS as an analytical tool.


Pharmaceutical Technology
Volume 34, pp. s12-s17

Brief history of H/DX–MS

Hydrogen exchange (HX) is a phenomenon that was identified in the 1950s by Hvidt and Linderstrom–Lang (13) and was used in developing the technique of hydrogen/deuterium exchange (H/DX). Later developments in H/DX coupled this technology with various spectroscopic techniques, which included NMR and FTIR to help better study and understand protein structure (14, 15). The eventual coupling of H/DX to MS (H/DX–MS) came in 1991 through the work of Katta et al. (16) and was further enhanced by the work of Zhongqi Zhang and David L. Smith while they were at Purdue University. They paired H/DX–MS experiments with proteolytic digestion (17). This latter union provides localized information concerning the relative amount and rate of H/DX in different parts of a protein. Such H/DX information can achieve peptide resolution as low as 5–10 amino–acid residues with nearly complete sequence coverage. H/DX–MS also has low sample requirements (down to a few picomoles) and can be performed in native or formulated buffer conditions. In addition, more recent advances in terms of both hardware and software now enable H/DX–MS experiments to be completed in a few days or less (12). As a result of these developments H/DX–MS has steadily gained in popularity as a technique for studying the higher–order structure of proteins.

The basics of H/DX–MS

The mechanisms of HX have been reviewed before in detail (18, 19). Briefly, HX occurs in a protein when labile hydrogen atoms change places with hydrogen atoms in solution. If an H2O solution is replaced with D2O (that is with deuterium instead of hydrogen), the labile hydrogen atoms in the protein will switch with deuterium atoms. In proteins, there are several different types of hydrogen atoms (e.g., aliphatic, hydroxyl, etc.), however, only the backbone amide hydrogen atoms exchange within a timeframe that can be monitored in a typical H/DX–MS experiment. Because every amino acid has a backbone amide (except for proline), these hydrogens act as sensors for monitoring the local conformational environment of the protein backbone (18). H/DX information can provide significant indirect insight into the conformational status of the protein polypeptide backbone. This information is especially informative in comparison studies of protein variants (19, 20).


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