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

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 data-dependent
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.