TY - JOUR
T1 - Graphitized carbon black enrichment and UHPLC-MS/MS allow to meet the challenge of small chain peptidomics in urine
AU - Piovesana, Susy
AU - Capriotti, Anna Laura
AU - Cerrato, Andrea
AU - Crescenzi, Carlo
AU - La Barbera, Giorgia
AU - Laganà, Aldo
AU - Montone, Carmela Maria
AU - Cavaliere, Chiara
N1 - CURIS 2019 NEXS 339
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Faculty of Science
KW - Peptides
KW - Peptide identification
KW - Biomarker discovery
UR - http://www.scopus.com/inward/record.url?scp=85071788629&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.9b03034
DO - 10.1021/acs.analchem.9b03034
M3 - Journal article
C2 - 31418265
AN - SCOPUS:85071788629
SN - 0003-2700
VL - 91
SP - 11474
EP - 11481
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
IS - 17
ER -