Digest Tool for automated detection of digest peptides in spectra. A protein sequence can be loaded and digested in silico in QUDeX-MS, and digest fragments can be matched against ions detected in spectra in order to better automate data analysis in a typical HDX experiment. Protein sequences can be pasted into the Protein Sequence field, loaded from a fasta file, or retrieved from NCBI using an appropriate accession. Cleavage specifities for many common proteolytical enzymes are included in QUDeX-MS. Custom cleavage definitions can also be specified, or all possible peptide fragments can be generated for a specified range of peptide lengths. Digest peptides can be added directly to the Command Window, further edited individually to add modifications, or can be searched against single or multiple spectra before adding to the Command Window. Digest peptides detected in loaded spectra are displayed in a results window (bottom panel). The mass error tolerance can be adjusted in this window, which will update match results automatically. Matching of digest peptides to ions detected in spectra is initially performed based on proper assignment of the monoisotopic mass. However, digest peptides can also be matched against distributions lacking a detectable monoisotopic peak by selecting the “Consider monoisotopic m/z not detectable?” check box. Peptides are then matched to isotopic distributions that fall within m/z bins associated with deuterium exchange-associated m/z’s within a reasonable distance from the monoisotopic mass (determined by the total number of deuterium exchangeable sites).