Improved Protein Hydrogen/Deuterium Exchange Mass Spectrometry Platform with Fully Automated Data Processing

Abstract

Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

“…We find that the effort to achieve residue resolution is not limited by a difficult calculational barrier (17,18,20) but by experimental difficulties. To reach this goal, it is necessary to obtain many sequentially overlapping peptide fragments that cover the experimental protein several times over.…”

“…However, in the absence of a common terminus, the D occupancy of the overhanging residues cannot be obtained by simple subtraction of the D-dependent mass centroids of neighboring peptides, as for the ECD/ETD dataset. Site-resolution analysis must consider simultaneously the multiple distributed segments and overhangs in the entire peptide collection (15,(17)(18)(19)(20). We find that this kind of analysis can be greatly improved by the use of an aspect of the MS data that has been ignored before, namely the multiple peaks in the isotopic envelope of each peptide fragment rather than just the single parameter mass centroid.…”

“…Decreased mass resolution has no effect on HDsite performance so long as the position and amplitude of isotopic peaks can be specified, although low resolution reduces the number of peptides able to be found. High levels of back exchange (>20%) can be a problem, and other workers have controlled for sizeable back exchange and for run-to-run variance by monitoring internal reference peptides (20). We have described methods for minimizing back exchange, and find run-to-run variance in back exchange to be small (<4%), as for experimentally measured D labeling in general.…”

“…More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Recently reported efforts have moved in this direction (15,(17)(18)(19)(20)(21)(22)(23)(24).…”

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

“…Hydrogen/deuterium exchange (HDX) MS has been widely used in the biopharmaceutical industry for higher-order structure characterization of protein therapeutics because of a welldeveloped automatic platform and the commercial availability of data analysis software [10][11][12][13][14][15][16][17]. By monitoring protein amide backbone HDX, which depends on the protein local solvent accessibility and hydrogen bonds, one can characterize the solution conformation and dynamics of proteins, including changes due to the external environment [18] (e.g., chemical modifications [19][20][21][22], buffer components [23,24], and the presence of a binding protein/ligand [25,26]).…”