Untargeted characterization of dansyl-labeled short peptides up to four amino acids in protein hydrolysates by LC-MS/MS

Proteins hydrolysates are complex mixtures with a diverse molecular composition, ranging from amino acids to short peptides, oligopeptides up to the truncated form of the proteins. Short peptides, especially those whose length extends up to 4 amino acids, are growingly gaining interest and their analysis in complex hydrolyzed matrices is crucial, knowing their exceptional nutritional value, organoleptic properties, and health-promoting features. If the analysis of amino acids is well established, the analysis of short peptides is still challenging due to their small size and extreme physico-chemical features, making their comprehensive LC-based separation and their MS-based detection tricky. Moreover, they are not covered by classical omics workflows. Herein, we present a workflow for the untargeted characterization of short peptides in proteins hydrolysates. Our developed method consists of an amino-group labeling of short peptides in lyophilized samples with dansyl chloride at basic pH. Following reaction quench and filtration, nanoESI nanoLC-MS/MS analysis is performed. The raw data was processed and treated with our in house-developed program in python language, short_pept, allowing the reliable identification of dansyl-labeled short peptides sequences with respective signal intensities and retention times. Chemical modifications such as methionine oxidation were successfully detected. Thanks to this workflow, we were able to characterize short peptides in various hydrolysates derived from soybean, casein, and yeast extracts and to classify them based on their degree of hydrolysis. This workflow aims to deeply understand the composition of hydrolyzed products, that varies widely depending on the starting raw material and hydrolysis process.