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Analytical Chem JoVE (Open Access) Citable · DOI

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

DOI: 10.3791/52503-v
What you'll learn
  • ✓ Apply ssHDX-MS to measure protein backbone dynamics in lyophilized powders
  • ✓ Use ssPL-MS to map protein surface exposure and conformational states
  • ✓ Interpret mass spectrometry data for formulation design and stability assessment
Protocol

Biopharma Insights Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Difficulty
advanced
Total time
~2–4 hours per sample (excluding MS instrument time and data processing)

Steps

1
Perform solid-state HDX-MS on intact proteins

Execute hydrogen/deuterium exchange on lyophilized protein powders and analyze intact molecular ions by mass spectrometry to assess overall backbone solvent accessibility and conformational dynamics.

▶ 01:39
2
Conduct peptide-level ssHDX-MS analysis

Digest exchanged protein samples into peptides and perform liquid chromatography-mass spectrometry to obtain residue-level deuterium incorporation, providing detailed backbone exposure information.

▶ 04:34
3
Apply photolytic labeling to intact proteins

Expose lyophilized protein powders to ultraviolet light to cross-link accessible side chains, then analyze intact mass shifts to reveal surface exposure patterns in the solid state.

▶ 06:23
4
Perform peptide-level photolytic labeling analysis

Digest photolabeled proteins and use mass spectrometry to map modified residues at amino acid resolution, identifying specific regions of protein-protein or protein-formulation interactions.

▶ 07:36
5
Extract backbone and side-chain exposure data

Integrate ssHDX-MS and ssPL-MS results to generate comprehensive exposure maps for protein formulations, supporting structural interpretation and formulation stability predictions.

▶ 09:24
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