pr9b00516_si_001.pdf (307.47 kB)
Sequencing Orbitides by Acid-Mediated Ring Cleavage Followed by Tandem Mass Spectrometry
journal contribution
posted on 2019-10-09, 13:44 authored by Mark F. Fisher, Joshua S. MylneHomodetic
cyclic peptides have aroused interest because of their
pharmacological potential. Sequencing cyclic peptides is difficultEdman
degradation is not possible as there is no N-terminus, NMR requires
quantities that are 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.