Sequencing orbitides by acid-mediated ring cleavage followed by tandem mass spectrometry

Research output: Contribution to journalArticle

Abstract

Homodetic cyclic peptides have aroused interest because of their pharmacological potential. Sequencing cyclic peptides is difficult - Edman degradation is not possible as there is no N-terminus, NMR requires quantities hard to gather from native samples, and tandem mass spectrometry data are difficult to interpret due to the peptide ring opening at multiple points. Sequencing can be simplified by cleaving the peptide ring at a specific peptide bond. Partial acid hydrolysis is a possible solution, but to date sequencing by this method has only been demonstrated for linear peptides and cyclotides, which are larger cyclic peptides (~30 amino acids) with three disulfide bonds. This study tests whether partial acid hydrolysis could be used to aid sequencing of Cys-less cyclic peptides with fewer than ten amino acid residues. We show that, with the right combination of temperature and acid, ring cleavage occurs and offers relatively simple MS/MS spectra amenable to sequencing. We describe how this method was used in our recent study in which we sequenced annomuricatin D (cyclo-GHSIFPPIP) from seeds of the soursop, Annona muricata. We found that orbitides can be linearized for MS/MS sequencing by incubation with 1.2 M HCl at 90 °C for 15-20 min. This fast, economical sequencing method will be useful to those studying small cyclic peptides lacking disulfide bonds, which are commonly found in many organisms, especially plants.

Original languageEnglish
JournalJournal of Proteome Research
DOIs
Publication statusE-pub ahead of print - 30 Sep 2019

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Cyclic Peptides
Tandem Mass Spectrometry
Mass spectrometry
Acids
Annona
Peptides
Disulfides
Cyclotides
Hydrolysis
Amino Acids
Seed
Seeds
Nuclear magnetic resonance
Pharmacology
Degradation
Temperature

Cite this

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title = "Sequencing orbitides by acid-mediated ring cleavage followed by tandem mass spectrometry",
abstract = "Homodetic cyclic peptides have aroused interest because of their pharmacological potential. Sequencing cyclic peptides is difficult - Edman degradation is not possible as there is no N-terminus, NMR requires quantities hard to gather from native samples, and tandem mass spectrometry data are difficult to interpret due to the peptide ring opening at multiple points. Sequencing can be simplified by cleaving the peptide ring at a specific peptide bond. Partial acid hydrolysis is a possible solution, but to date sequencing by this method has only been demonstrated for linear peptides and cyclotides, which are larger cyclic peptides (~30 amino acids) with three disulfide bonds. This study tests whether partial acid hydrolysis could be used to aid sequencing of Cys-less cyclic peptides with fewer than ten amino acid residues. We show that, with the right combination of temperature and acid, ring cleavage occurs and offers relatively simple MS/MS spectra amenable to sequencing. We describe how this method was used in our recent study in which we sequenced annomuricatin D (cyclo-GHSIFPPIP) from seeds of the soursop, Annona muricata. We found that orbitides can be linearized for MS/MS sequencing by incubation with 1.2 M HCl at 90 °C for 15-20 min. This fast, economical sequencing method will be useful to those studying small cyclic peptides lacking disulfide bonds, which are commonly found in many organisms, especially plants.",
author = "Fisher, {Mark F} and Mylne, {Joshua S}",
year = "2019",
month = "9",
day = "30",
doi = "10.1021/acs.jproteome.9b00516",
language = "English",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",

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TY - JOUR

T1 - Sequencing orbitides by acid-mediated ring cleavage followed by tandem mass spectrometry

AU - Fisher, Mark F

AU - Mylne, Joshua S

PY - 2019/9/30

Y1 - 2019/9/30

N2 - Homodetic cyclic peptides have aroused interest because of their pharmacological potential. Sequencing cyclic peptides is difficult - Edman degradation is not possible as there is no N-terminus, NMR requires quantities hard to gather from native samples, and tandem mass spectrometry data are difficult to interpret due to the peptide ring opening at multiple points. Sequencing can be simplified by cleaving the peptide ring at a specific peptide bond. Partial acid hydrolysis is a possible solution, but to date sequencing by this method has only been demonstrated for linear peptides and cyclotides, which are larger cyclic peptides (~30 amino acids) with three disulfide bonds. This study tests whether partial acid hydrolysis could be used to aid sequencing of Cys-less cyclic peptides with fewer than ten amino acid residues. We show that, with the right combination of temperature and acid, ring cleavage occurs and offers relatively simple MS/MS spectra amenable to sequencing. We describe how this method was used in our recent study in which we sequenced annomuricatin D (cyclo-GHSIFPPIP) from seeds of the soursop, Annona muricata. We found that orbitides can be linearized for MS/MS sequencing by incubation with 1.2 M HCl at 90 °C for 15-20 min. This fast, economical sequencing method will be useful to those studying small cyclic peptides lacking disulfide bonds, which are commonly found in many organisms, especially plants.

AB - Homodetic cyclic peptides have aroused interest because of their pharmacological potential. Sequencing cyclic peptides is difficult - Edman degradation is not possible as there is no N-terminus, NMR requires quantities hard to gather from native samples, and tandem mass spectrometry data are difficult to interpret due to the peptide ring opening at multiple points. Sequencing can be simplified by cleaving the peptide ring at a specific peptide bond. Partial acid hydrolysis is a possible solution, but to date sequencing by this method has only been demonstrated for linear peptides and cyclotides, which are larger cyclic peptides (~30 amino acids) with three disulfide bonds. This study tests whether partial acid hydrolysis could be used to aid sequencing of Cys-less cyclic peptides with fewer than ten amino acid residues. We show that, with the right combination of temperature and acid, ring cleavage occurs and offers relatively simple MS/MS spectra amenable to sequencing. We describe how this method was used in our recent study in which we sequenced annomuricatin D (cyclo-GHSIFPPIP) from seeds of the soursop, Annona muricata. We found that orbitides can be linearized for MS/MS sequencing by incubation with 1.2 M HCl at 90 °C for 15-20 min. This fast, economical sequencing method will be useful to those studying small cyclic peptides lacking disulfide bonds, which are commonly found in many organisms, especially plants.

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