Reversible Lysine Derivatization Enabling Improved Arg-C Digestion, a Highly Specific Arg-C Digestion Using Trypsin

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Bottom-up proteomics relies heavily on enzymatic digestion for peptide generation.
• Endopeptidase Arg-C offers potential for specific peptide bond cleavage but is limited by low specificity and high cost.

Purpose of the Study:

• To optimize a reversible amine derivatization method for improved Arg-C digestion in bottom-up proteomics.
• To evaluate the performance of the enhanced Arg-C (iArg-C) digestion compared to conventional methods.

Main Methods:

• Developed and optimized a reversible amine derivatization (citraconylation/decitraconylation) strategy.
• Applied this method in conjunction with trypsin digestion to create the iArg-C digestion protocol.
• Compared iArg-C digestion with conventional Arg-C, trypsin, and Lys-C digestion using peptide identification and cleavage specificity metrics.

Main Results:

• iArg-C digestion identified 64.2% more peptides than conventional Arg-C digestion (11,925 ± 199 vs 7262 ± 59).
• Achieved significantly higher cleavage specificity with iArg-C (95.6%) compared to conventional Arg-C (73.6%).
• iArg-C performance was comparable to Lys-C digestion and slightly better, though not as effective as trypsin digestion.

Conclusions:

• The optimized iArg-C digestion method significantly enhances peptide identification and specificity in bottom-up proteomics.
• iArg-C digestion represents a valuable and promising alternative proteomic strategy, potentially serving as a prior option to trypsin for increased proteome coverage.
• This method provides a robust approach for advancing proteomic research and data acquisition.