Untargeted Analysis of Short-Chain Peptides in Urine Samples Short Peptides Analysis

: Short-chain peptides have attracted increasing attention in different research fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices due to their low abundance compared to other molecules, which can cause extensive ion suppression during mass spectrometric acquisition. Moreover, there is a lack of analytical workflows for their comprehensive characterization since ordinary peptidomics strategies cannot identify them. In this context, an enrichment strategy was introduced and developed to isolate and clean up short-chain peptides by graphitized carbon black solid phase extraction. For better coverage of peptide polarity, urine samples were analyzed by ultrahigh performance liquid chromatography by reversed-phase and hydrophilic interaction liquid chromatography. High-resolution mass spectrometry allowed the detection of the eluting peptides by data-dependent mode using a suspect screening strategy with an inclusion list; peptides were identified by a semiautomated workflow implemented on Compound Discoverer. The complementarity of the orthogonal separation strategy was confirmed by peptide identification, resulting in 101 peptides identified from the RP runs, and 111 peptides from the HILIC runs, with 60 common identifications.