Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics

Area of Science:

• Proteomics
• Enzymatic Digestion
• Mass Spectrometry

Background:

• Complete enzymatic digestion is crucial for shotgun proteomics.
• Incomplete digestion limits proteome coverage and increases variability.
• Existing methods struggle with complete cleavage at arginine residues.

Purpose of the Study:

• To characterize Arg-C Ultra and Lys-C proteases for optimal sequential digestion.
• To develop a robust sequential digestion workflow compatible with label-free DIA and TMTpro multiplexing.
• To improve proteome coverage and quantitative reproducibility in proteomics.

Main Methods:

• Systematic characterization of Arg-C Ultra and Lys-C using chromogenic substrates and shotgun proteomics.
• Optimization of reaction conditions including temperature, pH, and reducing agents.
• Development and application of a sequential Lys-C/Arg-C Ultra digestion protocol.

Main Results:

• Arg-C Ultra is optimal at room temperature under reducing conditions; Lys-C is active at 37°C but sensitive to reductants.
• 1 mM TCEP preserves TMTpro integrity while enabling Arg-C Ultra activity.
• The developed protocol achieved >99% arginine and 95% lysine cleavage efficiencies.
• Quantified proteins increased by 6% (label-free DIA) and 11% (TMTpro).

Conclusions:

• A synergistic Lys-C/Arg-C Ultra digestion strategy enhances proteome coverage.
• The protocol is compatible with both label-free DIA and TMTpro multiplexing.
• This method provides excellent quantitative reproducibility for proteomics.