DIGging into the DIGestion

Protein digestion is an essential tool in both classical bottom-up proteomics and structural proteomics approaches such as chemical cross-linking, radical labelling or hydrogen/deuterium exchange. While proteomics uses specific proteases with predictable digestion patterns and minimal missed cleavages, HDX relies on non-specific or semi-specific acidic proteases. These are highly promiscuous in terms of amino acid preferences and tend to generate a lot of overlaps. Although this hampers predictability and adds complexity, it also has several advantages, such as a higher number of experimental data points and increased spatial resolution. As more proteolytic columns become available, the need for rigorous methods to compare digestion results increases. To meet these needs, we have developed a Java-based software tool that takes the exported raw search results (e.g. csv from MASCOT) and the corresponding database and performs an instant extraction of the key digestion parameters. This tool, called DigDig, provides digestion metrics, reproducibility assessment, coverage maps, extraction of cleavage preferences up to the N-th position, peptide length distribution, missed cleavage analysis and data export for IceLogo. It works with single proteins or complex mixtures, where it can plot data for either selected protein(s) or the entire identified ensemble. We will show how this software has helped us to identify novel cleavage preferences, optimise the HDX-MS workflow, but also its applicability in the evaluation of specific proteases, the key tool of the proteomic pipeline.

Financial support from the MEYS/EU OP JAK project PHOTOMACHINES - CZ.02.01.01/00/22_008/0004624 is gratefully acknowledged.