Revealing Formylation Sites in Intact Amphibian Disulfide Peptides: A Top-Down Study Using ExD.

Abstract

Mass spectrometry contributed significantly to the creation of proteomics as a science and is now the principal analytical tool of proteomics. Although the bottom-up approach remains the most popular, more and more studies apply the top-down method for sequencing. Novel tandem mass spectrometry methods resolved numerous sequencing problems and allowed for the analysis of intact peptides and even proteins. Natural frog peptides are convenient models to develop mass spectrometry methods for de novo sequencing of intact biomolecules. Here we demonstrate the ability of ExD tandem mass spectrometry to sequence intact peptides from skin secretions of Pelophylax ridibundus from the Rostov (Russia) population. Studying that secretion, we detected an array of mono- and diformylated skin peptides at various sites of the backbone. The main goal of the study dealt with the determination of the formylation sites of these up to 46-mer long peptides. ExD reliably solved the problem without any additional mass spectrometry experiments or preliminary chemical derivatization. We propose a mechanism for the observed formylation and a biochemical and experimental rationale.