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Anal Chem. 2012 Jun 5;84(11):4942-9. doi: 10.1021/ac300535r. Epub 2012 May 15.

Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing.

Author information

1
Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States. zzhang@amgen.com

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.

PMID:
22571272
DOI:
10.1021/ac300535r
[Indexed for MEDLINE]

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