ExD vs EThcD: What’s Better for the Direct Sequencing of Endogenous Amphibian Disulfide Peptides

Intact amphibian skin peptides, apart from their intrinsic interest, are a challenging model system to demonstrate direct sequencing, avoiding any preliminary derivatization steps. They are relatively long (up to 46-mer), contain an intramolecular disulfide bridge, and include a number of isomeric Leu/Ile residues. Sixteen intact peptides from the skin secretion of the Rostov (Russia) population of <i>Pelophylax ridibundus</i> were studied in the present research using EThcD, ExD, and ExciD fragmentation. Comparison of the efficiency of EThcD and ExD tandem mass spectrometry approaches demonstrated that both are appropriate for the direct sequencing of these peptides. Although the majority of the isomeric Leu/Ile residues could be differentiated using <i>w</i>-ions, the usefulness of <i>d</i>-ions, especially inside C-terminal disulfide rings, was also demonstrated. The <i>d</i>-ions arise more often in ExD/ExciD than in the EThcD mode. EThcD and ExciD are complementary methods and together distinguished more isomeric residues than either of them alone. While both methods provided similar sequence information within intact C-terminal S-S loops, their combined use consistently yielded 100% sequence coverage. ExciD demonstrated superior results in determining peptide sequences due to the higher yield of all fragment ion types, establishing complete sequences for all peptides, including that of the longest (46-mer) esculentins, compared to six with EThcD alone. The increased number of characteristic ions (<i>c/z</i> and <i>b/y</i>) in ExciD further enhanced confidence in the sequence assignments. Ultimately, the complementary use of ExciD and EThcD resulted in reliable 100% sequence coverage for all 16 intact disulfide peptides analyzed in this study.