A hydrogen-deuterium exchange mass spectrometry-based protocol for protein-small molecule interaction analysis

Area of Science:

• Biochemistry
• Structural Biology
• Analytical Chemistry

Background:

• Protein-small molecule interactions are crucial for cellular processes.
• Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a key technique for studying these interactions.
• Optimizing HDX-MS experimental conditions is essential for accurately probing conformational dynamics.

Purpose of the Study:

• To present a detailed continuous-labeling, bottom-up HDX-MS protocol.
• To demonstrate the protocol's utility in studying protein-small molecule interactions.
• To facilitate structural characterization of these interactions for researchers.

Main Methods:

• Development and application of a continuous-labeling, bottom-up HDX-MS protocol.
• Side-by-side HDX kinetics comparison of Hsp90N.
• Treatment of Hsp90N with small molecules (Radicicol, Geldanamycin).

Main Results:

• The protocol successfully displayed conformational changes in Hsp90N induced by small molecule binding.
• HDX kinetics were compared for Hsp90N with and without small molecule treatment.
• The method proved sensitive and robust for structural characterization.

Conclusions:

• The presented HDX-MS protocol is effective for studying protein-small molecule interactions.
• This method allows for the sensitive and robust structural characterization of conformational changes.
• The protocol is accessible to researchers for investigating molecular interactions.