A new opening for the tricky untargeted investigation of natural and modified short peptides.

Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose. First, they were employed as inclusion lists for a suspect screening mass-spectrometric analysis, overcoming the limits of data dependent acquisition mode and allowing the fragmentation of such low-abundance substances. Moreover, the databases were implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, for the creation of a data processing workflow specifically dedicated to short peptide tentative identification. For this purpose, a detailed study of short peptide fragmentation pathways was carried out for the first time. The proposed method was applied to the study of short peptide sequences in enriched urine samples and led to the tentative identification more than 200 short natural and modified short peptides, the highest number ever reported.