Rheostatic control of tryptic digestion in a microscale fluidic system

Area of Science:

• Proteomics and analytical chemistry
• Biochemistry and molecular biology
• Microfluidics and systems engineering

Background:

• Complete protein characterization requires integrating bottom-up and top-down proteomic strategies.
• Current blended proteomic approaches necessitate fraction collection, complicating the workflow.
• A flow-through system capable of regulating digestion efficiency is needed to streamline proteomic analysis.

Purpose of the Study:

• To investigate the regulation of tryptic digestion within an immobilized enzyme reactor (IMER) using mixed solvent systems.
• To develop a method for controlling digestion efficiency in a closed fluidic system for proteomic analysis.
• To demonstrate the feasibility of blending bottom-up and top-down proteomics in a single, integrated system.

Main Methods:

• Utilized an immobilized enzyme reactor (IMER) integrated with a dual gradient pumping system and mass spectrometer.
• Employed programmable solvent waveforms to oscillate acetonitrile concentration (0-45%) in the fluid stream.
• Tested the system using protein standards: ovalbumin, cytochrome c, and myoglobin.

Main Results:

• Demonstrated efficient regulation of tryptic digestion from complete digestion to no digestion by modulating solvent composition.
• Observed novel peptide mass maps due to substrate unfolding hysteresis, alongside intact protein recovery.
• Achieved reproducible and stable results with cycle times around 90 seconds.

Conclusions:

• Regulated digestion in a closed fluidic system is achievable and efficient.
• This rheostatic digestion mode offers access to both intact proteins and unique peptide maps.
• The developed technique is suitable for integrating bottom-up and top-down proteomics, eliminating the need for fraction collection.