Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protease activity analysis is crucial in biological research and diagnostics.
• Existing methods for multiplex protease analysis can be complex and lack sensitivity.
• MALDI-TOF mass spectrometry offers high throughput and sensitivity for molecular analysis.

Purpose of the Study:

• To develop a peptide-encoded microplate for simultaneous identification and activity analysis of multiple proteases.
• To establish a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry-based method for protease detection.
• To validate the assay using model proteases like trypsin and chymotrypsin.

Main Methods:

• Design of peptide codes with a coding region and protease-susceptible substrate.
• Enzymatic cleavage of peptide codes by target proteases.
• Quantification of released coding regions ('Protease ID') using MALDI-TOF MS with internal standards.
• Verification using trypsin and chymotrypsin as model proteases.

Main Results:

• The peptide-encoded microplate successfully identified 'Trypsin ID' and 'Chymotrypsin ID' at specific m/z values.
• Logarithmic intensity ratios of 'Protease ID' to internal standards correlated with protease concentrations (5.0–500 nM for trypsin, 10–500 nM for chymotrypsin).
• Achieved detection limits of 2.3 nM for trypsin and 5.2 nM for chymotrypsin, demonstrating high sensitivity and selectivity.

Conclusions:

• The peptide-encoded microplate is a powerful tool for convenient multiplex protease identification and activity analysis.
• This method offers a sensitive, selective, and high-throughput approach for studying protease function.
• The assay has potential applications in biochemical research, drug discovery, and diagnostics.