Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Shotgun proteomics relies on efficient proteolysis for sensitive peptide and protein analysis.
• Sequential LysC/trypsin digestion is reported to yield more fully tryptic peptides than single-tryptic methods.
• Previous studies assumed optimal tandem proteolysis required high sample denaturation.

Purpose of the Study:

• To investigate LysC/trypsin tandem digestion efficiency in Mass Spectrometry-compatible detergents.
• To compare tandem digestion with sole trypsin digestion.
• To assess the impact of tandem digestion on absolute protein quantification.

Main Methods:

• Comparison of sole trypsin digestion with LysC/trypsin tandem digestion.
• Proteolysis performed in Mass Spectrometry-compatible detergents and chaotropic urea.
• Evaluation of peptide recovery and absolute protein quantification using intensity-based absolute quantification (iBAQ) values.

Main Results:

• LysC/trypsin tandem digestion achieved high efficiency in detergents, outperforming sole trypsin.
• Tandem digestion in detergents yielded higher quantitative recovery of fully cleaved peptides.
• Increased cleavage efficiency positively impacted iBAQ values for absolute protein quantification.
• The benefits of LysC/trypsin digestion are attributed to enzyme specificity, not sample denaturation.

Conclusions:

• Tandem LysC/trypsin proteolysis is effective in Mass Spectrometry-compatible detergents, enhancing peptide yield and protein quantification.
• This detergent compatibility allows integration into cellular solubilization proteomics workflows, avoiding buffer exchange.
• The findings suggest enzyme specificity, rather than denaturation, drives the advantages of sequential digestion.