Accepted: Hans McCullagh Lipids

Introduction

Lipid signaling analytes represent a diverse group of biomolecules that have essential roles in structural, storage and signaling processes in living systems. Class separation is readily achieved using chromatographic and MS based identification techniques; however, the analysis remains challenging due to the chemical structure diversity and isobaric nature of these types of compounds. The addition of IMS to discovery workflows enhances system peak capacity and improves isomer resolution. IM separation was achieved using a multi-pass travelling-wave cyclic IM (cIM)-device, where increasing the number of passes around the device allows of the increase in both mobility resolution and ion residence time. MS and CID fragmentation data were obtained on precursor IM separated analytes followed by ToF mass measurement.

Methods

Unsaturated free fatty acid (FA) standards, differing in chain length and number of cis/trans configurations, steroid hormones (androgens), isomers differing in the positon of a functional group, and isobaric lipid mediators (prostaglandins) were chosen to determine the degree of IM separation required to separate isomers and isobars. Data were collected on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. In all experiments, the isomeric and isobaric compounds were introduced as two or three component mixtures by means of direct introduction. Both precursor and product ion CCS data were collected, facilitated by a multifunction region within the cIM device that can switch function to inject and eject ions, respectively, following IM separation.

Preliminary Data

In all direct infusion cIM-MS measurements, cis oriented FAs were found to be more compact than those with trans-orientations. A different number of cycles through the cIM -device, thereby increasing the effective path length/resolution, were required to achieve a similar degree of IM separation for mono-unsaturated FAs of differing chain length. The required IM resolution (Ω/ΔΩ) values typically ranged from 100 to 350. Unsaturated FAs with two or more double bonds, separated by two mid-chain carbons, could not be distinguished. Shorter, structurally more rigid and compact FAs were discriminated at reduced resolution, as could longer chain mono-unsaturated FAs as a result of partial chain back-folding. Following IM separation, isomeric FAs were successfully CID fragmented and identified.

The analysis of 17-hydroxyprogesterone and 21-hydroxyprogesterone, an isobaric adrenal steroid pair, indicated that an IM resolution (Ω/ΔΩ) of at least 200 was required to baseline separate this lipid signal analytes class. Following IM separation, nearly identical, but individual product ion spectra were readily detected, arguing the need for the separation of these types of compounds. A three compound isomer steroid mixture consisting of 11-deoxycortisol, 21-deoxycortisol, and corticosterone was partially resolved. Here, after 12 passes through the cIM device, 21-deoxycortisol could be separated from the two other steroids, and following 20 passes, 11-deoxycortisol but with the two remaining steroids now partially resolved. Hence, complete separation of corticosterone could not be achieved. The IM separation was however sufficient to extract product ion spectra for the three individual components and establish their identity using informatics methodology.

The separation of other lipid signaling class analytes, such as prostaglandins and other lipid mediators, by cIM will also be investigated and presented.

Novel Aspect

A Q-cIM-oaToF research platform has been characterized and applied to the IM separation of isomeric lipid signaling analytes.

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Poster:

Ion Mobility: Applications

Submitting Author:

Hans Vissers
Waters Corporation
Wilmslow,
hans_vissers@waters.com