High-resolution H/D exchange studies on the HET-s218-295 prion protein

Area of Science:

• Biochemistry
• Structural Biology
• Mass Spectrometry

Background:

• Hydrogen/deuterium (H/D) exchange coupled with mass spectrometry (HXMS) is crucial for studying protein structure and solvent accessibility.
• Previous HXMS studies have faced limitations in achieving high spatial resolution for detailed structural insights.

Purpose of the Study:

• To evaluate two distinct methodologies for enhancing the resolution of HXMS experiments in analyzing the HET-s(218-295) prion protein.
• To investigate solvent accessibility and identify protected regions within the prion protein structure.

Main Methods:

• The first approach involved pepsin digestion of aggregated HET-s(218-295) followed by analysis of peptide deuterium incorporation using nanospray mass spectrometry with and without collision-induced dissociation (CID).
• The second approach analyzed deuterium incorporation across 76 peptides of the HET-s(218-289) sequence, utilizing shared boundaries for high-resolution regional deuteration analysis.

Main Results:

• The first method, using CID on an ion trap mass spectrometer, revealed significant H/D scrambling in fragment ions, limiting spatial resolution.
• The second method successfully determined deuteration levels for small regions (1-4 amino acids) by analyzing overlapping peptic peptides.
• This second approach provided evidence for the existence of highly protected regions within the HET-s(218-295) sequence.

Conclusions:

• Collision-induced dissociation (CID) in ion trap mass spectrometry is not suitable for improving spatial resolution in HXMS due to H/D scrambling.
• A refined HXMS methodology, analyzing overlapping peptides, enables precise determination of deuteration levels and identification of protected regions in prion proteins.