High-Resolution Mass Spectrometric Methods for Proteins in Lyophilized Solids

  • Karthik Balakrishna Chandrababu
  • Rajashekar Kammari
  • Yuan Chen
  • Elizabeth M. ToppEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Lyophilization (freeze-drying) is used to produce amorphous solid powders of protein drugs. Though lyophilization is usually used in an attempt to stabilize the protein, degradation processes can still occur in the solid state, and are often poorly correlated with measurable properties of the protein and the powder. This chapter describes two novel, high-resolution mass-spectrometry-based methods for assessing protein structure and interactions in solid powders: solid-state hydrogen-deuterium exchange (ssHDX) and solid-state photolytic labeling (ssPLL) with mass spectrometric analysis (ssHDX-MS, ssPLL-MS). ssHDX-MS measures the rate of deuterium incorporation in the protein on exposure of the solid powder to D2O vapor. ssHDX-MS is thought to provide information regarding the network of inter- and intramolecular hydrogen bonds experienced by the protein in the solid state, and recent studies have shown that ssHDX metrics are highly correlated with stability on storage. ssPLL-MS provides complementary information on the protein’s side chain environment. The chapter summarizes the methods and recent results both ssHDX-MS and ssPLL-MS, and suggests directions for future research.

Key words

Mass spectrometry Hydrogen-deuterium exchange (HDX) Solid-state HDX Photolytic labeling Peptides Proteins 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Karthik Balakrishna Chandrababu
    • 1
  • Rajashekar Kammari
    • 1
  • Yuan Chen
    • 1
  • Elizabeth M. Topp
    • 1
    Email author
  1. 1.Department of Industrial and Physical PharmacyPurdue UniversityWest LafayetteUSA

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