Single-day protein LC-MS bioanalysis: can next-generation trypsins cut it?

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Increasing demand for sensitive LC-MS assays has led to lengthy and complex methods.
• Simplifying protein digestion protocols is crucial for improving throughput and efficiency in LC-MS analysis.

Purpose of the Study:

• To evaluate next-generation trypsin enzymes for protein digestion in LC-MS.
• To identify a trypsin that can simplify LC-MS method development and increase sample throughput.

Main Methods:

• Assessed commercial next-generation trypsins using protein standards in buffer and complex matrices.
• Utilized liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for performance evaluation.

Main Results:

• All tested next-generation trypsins outperformed overnight tryptic digestion, achieving comparable results in less time.
• Heat-stable trypsins may benefit from pre-digestion reduction and alkylation steps, warranting further investigation.

Conclusions:

• Promega Rapid-Digestion Trypsin demonstrated the best performance among the next-generation trypsins evaluated.
• This enzyme surpasses traditional overnight tryptic digestion, offering a more efficient alternative for LC-MS workflows.