Optimization of a Hydrogen Exchange-Mass Spectrometry Robotic Liquid Handler Using Tracers

Area of Science:

• Analytical Chemistry
• Biochemistry
• Protein Science

Background:

• Hydrogen exchange-mass spectrometry (HX-MS) is vital for studying protein structure and interactions.
• Achieving high reproducibility in HX-MS is challenging due to variability in deuterium labeling processes.
• Inconsistent solution dispensing, transfer, and mixing significantly impact experimental precision.

Purpose of the Study:

• To address the poor precision observed in robotic liquid handler operations for HX-MS.
• To develop and validate a method for optimizing HX-MS workflow precision.
• To provide insights into liquid handler performance for improved experimental reproducibility.

Main Methods:

• Utilized caffeine, caffeine-d3, and caffeine-d9 as tracers for sample, label, and quench steps.
• Tracked tracer performance to monitor and report on each liquid handling operation.
• Analyzed tracer data to identify sources of variability in the HX-MS workflow.

Main Results:

• Successfully improved the precision of the robotic liquid handling system for HX-MS.
• Demonstrated the effectiveness of the three-tracer approach in identifying performance issues.
• Established a method for real-time performance monitoring of liquid handling operations.

Conclusions:

• The three-tracer method significantly enhances the precision of HX-MS workflows.
• This approach aids in optimizing both manual and automated liquid handling in HX-MS.
• Tracers can be integrated into experiments for quality control and potential uptake correction.