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Protein Digestion01:02

Protein Digestion

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Protein digestion begins in the stomach, where the highly acidic environment can easily disrupt protein structure by exposing the peptide bonds of polypeptide chains. After polypeptide chains are broken into individual amino acids by a series of digestive enzymes, the amino acids are transported to the liver via the bloodstream to produce energy.
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DigDig: A Software for In-Depth Analysis and Comparison of Proteolytic Digestion.

Zuzana Kalaninová1,2, Jasmína Mária Portašiková1,2, Daniel Kavan1,2

  • 1Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, Prague 2, Prague 12843, Czech Republic.

Analytical Chemistry
|September 25, 2025
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Summary

DigDig software analyzes proteolytic digestion for mass spectrometry, improving peptide identification and sequence coverage in proteomics. It offers detailed visualizations for quality control and method development, especially for hydrogen/deuterium exchange mass spectrometry (HDX-MS).

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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.