Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine

Abstract

Short peptide sequences represent emerging analytes in a variety of fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices, due to the low abundance, extensive suppression during MS analysis, and lack of workflows, as they cannot be identified by ordinary peptidomics strategies and coverage is extremely limited by metabolomics as well. In this context, in this work, a solid phase extraction method was developed for the cleanup and enrichment of dipeptides, tripeptides, and tetrapeptides in urine using graphitized carbon black Carbograph 4 as the sorbent. The method was first developed on analytical standards spiked in urine, with recoveries in the range of 60−100%. Then the method was applied to urine samples from healthy volunteers. The enriched urine samples were analyzed by ultrahigh performance liquid chromatography (UHPLC) using an orthogonal strategy in which both a reversed phase (RP) C18 column and a zwitterionic hydrophilic interaction liquid chromatography (HILIC) column were used, for better coverage of peptide polarity and improved detection of peptides. High-resolution mass spectra were acquired in datadependent mode using a suspect screening strategy with inclusion list; peptides were identified by a semiautomated workflow for feature extraction, candidate mass filtering, and MS/MS spectra comparison with in silico mass spectra. 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. The method is applicable to both hydrophobic and hydrophilic peptides. Peptides were stable over 2 h after collection and protease inhibitors were not necessary, as no formation of artifacts was observed.

Identification and Antimicrobial Activity of Medium-Sized and Short Peptides from Yellowfin Tuna (Thunnus albacares) Simulated Gastrointestinal Digestion

“…Short-sized peptide purification was obtained thanks to a specific protocol which was previously optimized in our previous work on a GCB sorbent [ 21 ]. After washing to remove the impurities from the cartridge, the GCB sorbent was first activated with 0.1 mol L −1 HCl and then conditioned with 20 mmol L −1 TFA.…”

“…Regarding small peptides (2–4 amino acids long), at present, only two studies reported the identification of two antimicrobial tripeptides (IQY and YVL) and two antimicrobial tetrapeptides (EIPT and CIRA) generated by the enzymatic hydrolysis of bovine lactoferrin and kappa casein. The small number of publications on short AMPs is mainly ascribed to the several issues in the separation and identification of small amino acidic sequences [ 19 , 20 , 21 , 22 ].…”

Peptidome: Chaos or Inevitability

“…This could reflect biological trends in the composition of cellular and secretome peptidomes or be a result of biases in sample preparation and LC-MS/MS analysis. For example, very hydrophilic short peptides can be lost during C18 separation [ 115 ].…”

High-Resolution Mass Spectrometry and Chemometrics for the Detailed Characterization of Short Endogenous Peptides in Milk By-Products

“…For assessing the short peptide profile of milk byproducts derived from the cheesemaking industry, whole milk (WM), whole whey (WW), skimmed whey (SW), and permeated whey (PW) were purified with graphitized carbon black (GCB) [25] and analyzed by HPLC coupled to HRMS using a suspect screening approach. A database of all combinations of the 20 natural a.a. in di-, tri-, and tetrapeptides was obtained by MATLAB, filtered to remove repeating masses, and employed to generate a list of m/z to be implemented as an inclusion list in the MS method.…”

“…Samples were then centrifuged at 8000× g for 35 min at 4 • C to remove any debris. The supernatants were purified on Carbograph 4 cartridges using a previously developed protocol for short peptide purification and pre-concentration [25]. Briefly, the cartridge was first washed to remove impurities, then activated with 10 mL of H 2 O 0.1 mol L −1 HCl and conditioned with 10 mL of H 2 O 20 mmol L −1 TFA.…”