Accepted: Vanderbilt McLean

Introduction

Vasotocin nonapeptides are 9-amino acid peptide hormones responsible for fluid regulation (vasopressin family) and promoting social bonding (oxytocin family) and are implicated in a variety of developmental disorders, including autism, depression, and schizophrenia. The mechanism of action involves binding to vascular receptors, and a synthetic analogue, desmopressin, has demonstrated significantly decreased binding affinity through the substitution of the 8th residue, L-arginine, with its enantiomer, D-arginine, suggesting this residue significantly impacts the resulting peptide conformation. Prior ion mobility-mass spectrometry (IM-MS) studies showed direct IM separation of chiral nonapeptides. Here we present results from IM-MS and cyclic traveling wave IM (cIM) that resolve additional conformer populations within these diastereomeric peptides, as well as evidence for interconverting conformers co-existing during IM-MS analyses.

Methods

Samples of achiral pressinoic acid, and each diastereomer of the chiral compounds oxytocin, vasopressin, and desmopressin along with 1:1 mixtures of the diastereomeric pairs were prepared at a concentration of 10 μg mL−1 in ultra-pure water with 10 mM ammonium acetate added (pH ~6.5). Ion mobility separations and collision cross section (CCS) measurements were performed on a commercial drift tube IM-MS (Agilent) with IM resolutions of ~50. High resolution IM separations were performed on a cyclic traveling wave IM-MS (cIM) instrument (Waters) with accessible resolutions between 65 (1 cyclic pass) and >250 (18 passes). Additionally, tandem IM2 and IM3 experiments were carried out using cIM in a store/release mode whereby discrete IM populations are selected for subsequent IM analyses.

Preliminary Data

The diastereomers of vasopressin [H-Cys(1)-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D/L-Arg-Gly-NH2] and desmopressin [deamino-Cys(1)-Tyr-Phe-Gln-Asn-Cys(1)-Pro-D/L-Arg-Gly-NH2] are lasso peptides with a 6-amino acid ring created by a disulfide bridge (Cys1-Cys6) and 3-amino acid tail. Pressinoic acid [H-Cys(1)-Tyr-Phe-Gln-Asn-Cys(1)-OH] is an achiral hexapeptide ring that represents the ring scaffold for the vasopressins. IM-MS results for pressinoic acid yields two partially resolvable peaks under low resolution (~50) conditions, whereas high resolution cIM analysis (~200, 10 passes) reveals three baseline resolved conformer populations which, when individually selected for IM2 analyses, exhibited primarily a single IM population, suggesting these conformers are stable. For vasopressin, the [D-Arg8] diastereomer exhibits a single IM peak under both low (~50) and high resolution (~150) IM analyses, whereas [L-Arg8]-vasopressin exhibits two partially-resolvable peaks at low resolution (~50) which fail to resolve further at higher resolutions, but instead shows a “bridge” between the two peaks, indicating these two conformer populations are interconverting. Tandem IM2 and IM3 results all yield two distinct peaks which align in drift time to the precursor mobilities irrespective of which precursor peak is selected, providing further evidence of interconversion between the two conformer populations. Results were also obtained for oxytocin diastereomers [H-Cys(1)-Tyr-Ile-Gln-Asn-Cys(1)-Pro-D/L-Leu-Gly-NH2], which differs from the vasopressins by the identity of the 3rd and 8th residue. A mixture of the oxytocin diastereomers [D-Arg8] and [L-Arg8] are fully-resolved by cIM after 18 passes (~280 resolution), and exhibit only a single peak for tandem IM2 analyses of each diastereomer, suggesting that oxytocin expresses a narrow distribution of conformers. These results suggest that the 8th residue heavily influences the nonapeptide conformation. Preliminary computational results suggest these two conformer populations are an open and closed structure, the latter resulting from a strong ring-tail interaction. Initial metal cation studies aimed at disrupting these interactions have yielded complex IM spectra with multiple peak features.

Novel Aspect

Multiple conformer populations for lasso peptides revealed by high resolution cyclic IM and tandem IM/IM experiments.
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A post-doc is presenting author on this abstract? No
A graduate student is presenting author on this abstract? No
An undergraduate student is presenting author on this abstract? No

Oral Choice:

Ion Mobility: Structure
Second Oral Choice:

Ion Mobility: Small Molecules, Pharmaceuticals, and DMPK
Poster:

Ion Mobility: Fundamentals

Submitting Author:

John A. McLean
Vanderbilt University
Nashville, TN
john.a.mclean@vanderbilt.edu