Surrogate H/D detection strategy for protein conformational analysis using MS/MS data.

Area of Science:

• Proteomics
• Biophysical Chemistry
• Analytical Chemistry

Background:

• Amide hydrogen/deuterium exchange (H/DX) coupled with mass spectrometry (MS) is crucial for studying protein structure and dynamics.
• Existing MS methods face limitations in sensitivity and sequence coverage for complex multiprotein systems.

Purpose of the Study:

• To evaluate tandem mass spectrometry (MS/MS) as an alternative to conventional MS for bottom-up H/DX experiments.
• To determine if collisionally induced dissociation (CID)-generated product ions can effectively surrogate intact peptides for H/DX analysis.

Main Methods:

• Investigated deuterium measurements of calmodulin in apo and holo forms using peptic digests.
• Acquired H/DX data in both MS and MS/MS domains, analyzing % deuteration, fragment ion selection, and contaminant levels.
• Controlled isotopic envelope expansion (< or = 50% deuterium label) for MS/MS data acquisition.

Main Results:

• MS/MS data acquisition for deuterated peptides requires controlled isotopic envelope expansion.
• Fragment ions (excluding neutral loss) generally reflect apo/holo transition deuteration ratios similar to precursor ions.
• Base peak ion in MS/MS spectra showed the best correlation, benefiting from H/D scrambling under CID conditions.
• The surrogate approach successfully recovered accurate deuteration levels when conventional H/DX-MS was hindered by spectral interference.

Conclusions:

• CID-based automated H/DX-MS/MS acquisition provides a generalized strategy for analyzing complex protein systems.
• This approach extends the peptide capacity beyond conventional H/DX-MS.
• MS/MS offers an alternative dimension for H/DX analysis, enhancing sensitivity and sequence coverage.