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

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.