Retention Time Interpolation Enhances Peptide Mapping for HDX-MS.

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Proteomics

Background:

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is crucial for studying protein structural dynamics.
• Effective peptide mapping is essential for accurate HDX-MS analysis, requiring high sequence coverage and redundancy.
• Short liquid chromatography (LC) gradients in HDX-MS workflows limit peptide identification due to back-exchange constraints.

Purpose of the Study:

• To develop a flexible workflow that overcomes limitations of short LC gradients in HDX-MS.
• To enhance peptide identification, sequence coverage, and redundancy in HDX-MS experiments.
• To enable the use of rich peptide maps from long LC gradients without compromising deuterium retention.

Main Methods:

• Introduced a workflow using long LC gradients for initial peptide mapping, followed by retention time (RT) interpolation.
• Generated a regression model using shared peptides from long- and short-gradient mapping to predict short-gradient RTs.
• Implemented the method using 'RTinterpolator', a freely available R script compatible with HDX analysis platforms.

Main Results:

• The RTinterpolator workflow successfully predicts short-gradient RTs for peptides identified in long-gradient experiments.
• This approach allows leveraging comprehensive peptide maps from long gradients within the constraints of HDX-MS.
• Achieved increased sequence coverage and redundancy in HDX-MS data, particularly for complex protein samples.

Conclusions:

• RTinterpolator provides a practical and accessible solution for improving HDX-MS data quality.
• The method is instrument-independent and enhances the information content of HDX-MS studies.
• Facilitates more detailed analysis of protein structural dynamics, especially for challenging protein targets.