DigDig: A Software for In-Depth Analysis and Comparison of Proteolytic Digestion

Area of Science:

• Proteomics and Mass Spectrometry
• Bioinformatics and Computational Biology

Background:

• Proteolysis is essential for proteomics, impacting peptide identification and sequence coverage.
• Structural mass spectrometry (MS) methods like hydrogen/deuterium exchange MS (HDX-MS) use nonspecific proteases, generating complex peptide mixtures.
• Analyzing these complex digestions is critical for accurate results.

Purpose of the Study:

• To develop a software tool, DigDig, for evaluating and comparing proteolytic digestion.
• To streamline the analysis of complex peptide mixtures from diverse proteomic experiments.
• To enhance quality control and method development in proteomics, particularly for HDX-MS.

Main Methods:

• Developed DigDig, a standalone Java-based software.
• Processes output files from common proteomics search engines.
• Provides customizable visualizations of digestion metrics: sequence coverage, reproducibility, redundancy, cleavage sites, and peptide length.

Main Results:

• DigDig effectively evaluates proteolytic digestion across various conditions.
• The software visualizes key digestion metrics, aiding in quality control.
• A unique feature is the detection of repetitive peptide sequences often missed by other tools.

Conclusions:

• DigDig is a valuable tool for analyzing proteolytic digestion in proteomics workflows.
• It enhances digestion quality control, protease characterization, and method development.
• The software is particularly beneficial for complex HDX-MS experiments and is freely available.