Graphitized carbon black enrichment and UHPLC-MS/MS allow to meet the challenge of small chain peptidomics in urine

Susy Piovesana; Anna Laura Capriotti; Andrea Cerrato; Carlo Crescenzi; Giorgia La Barbera; Aldo Laganà; Carmela Maria Montone; Chiara Cavaliere

doi:10.1021/acs.analchem.9b03034

Graphitized carbon black enrichment and UHPLC-MS/MS allow to meet the challenge of small chain peptidomics in urine

Susy Piovesana, Anna Laura Capriotti^*, Andrea Cerrato, Carlo Crescenzi, Giorgia La Barbera, Aldo Laganà, Carmela Maria Montone, Chiara Cavaliere

^*Corresponding author for this work

Nutrition and Health

9 Citations (Scopus)

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 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.

Original language	English
Journal	Analytical Chemistry
Volume	91
Issue number	17
Pages (from-to)	11474-11481
Number of pages	8
ISSN	0003-2700
DOIs	https://doi.org/10.1021/acs.analchem.9b03034
Publication status	Published - 2019

Keywords

Faculty of Science
Peptides
Peptide identification
Biomarker discovery

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10.1021/acs.analchem.9b03034

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title = "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 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.",

keywords = "Faculty of Science, Peptides, Peptide identification, Biomarker discovery",

author = "Susy Piovesana and Capriotti, {Anna Laura} and Andrea Cerrato and Carlo Crescenzi and {La Barbera}, Giorgia and Aldo Lagan{\`a} and Montone, {Carmela Maria} and Chiara Cavaliere",

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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

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DO - 10.1021/acs.analchem.9b03034

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SN - 0003-2700

VL - 91

SP - 11474

EP - 11481

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 17

ER -

Graphitized carbon black enrichment and UHPLC-MS/MS allow to meet the challenge of small chain peptidomics in urine

Abstract

Keywords

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