Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry

Methods Mol Biol. 2023:2718:303-334. doi: 10.1007/978-1-0716-3457-8_17.


Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

Keywords: Antibody; Biosimilars; Hydrogen/deuterium exchange; Mass spectrometry; Protein structure and dynamics; Proteolysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies*
  • Deuterium
  • Hydrogen
  • Hydrogen Deuterium Exchange-Mass Spectrometry*
  • Mass Spectrometry


  • Deuterium
  • Antibodies
  • Hydrogen