Six alternative proteases for mass spectrometry-based proteomics beyond trypsin

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Trypsin is the standard protease for mass spectrometry (MS)-based shotgun proteomics.
• Sole reliance on trypsin limits proteome coverage, potentially missing post-translational modifications and protein subsets.
• Systematic documentation of alternative protease protocols is lacking.

Purpose of the Study:

• To provide an optimized, robust, and easy-to-use protocol for six alternative proteases.
• To enable parallel digestion experiments complementing trypsin.
• To offer guidance on protease selection, preparation, and MS data analysis.

Main Methods:

• Developed and optimized protocols for six alternative proteases: chymotrypsin, LysC, LysN, AspN, GluC, and ArgC.
• Evaluated protease availability, usage, and reagent preparation.
• Applied protocols to single protein (BSA) and complex cellular lysate (E. coli) digestion.
• Described appropriate MS data analysis methods.

Main Results:

• Presented a convenient and robust parallel digestion protocol for six alternative proteases.
• Provided data on protease characteristics and recommendations for use.
• Demonstrated applicability on both single protein and complex lysate samples.
• Protocol completion time is approximately 2 days.

Conclusions:

• The developed protocol enhances proteome coverage beyond trypsin limitations.
• Offers a standardized approach for utilizing alternative proteases in proteomics.
• Facilitates deeper proteome analysis and identification of post-translational modifications.