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Abstract INTRODUCTION:
Lipid mediators are cell-derived signaling molecules that modulate numerous biological functions. Of note, they play a significant role in all stages of inflammation, including its initiation, propagation, and resolution. Consequently, this class of molecules and their biochemical pathways are attractive candidates for anti-inflammatory drug development. Lipid mediators are potent signaling molecules whose endogenous concentrations are in the pM to nM range, making them a challenge to analyze. Typically, these molecules are measured with highly sensitive triple quadrupole mass spectrometers using multiple reaction monitoring (MRM) scan modes. However, the recent advent of an ultra-sensitive high-resolution mass spectrometer (HRMS) now enables accurate and precise measurements of lipid mediators in vivo while simultaneously providing critical structural characterization.
The ZenoTOF 7600 system is an HRMS instrument capable of measuring the low endogenous concentrations of many lipid mediators with a sensitivity level rivaling that of high-end triple quadrupole instruments. In addition to traditional fragmentation by collision-induced dissociation (CID), the instrument has a complementary fragmentation mode, electron-activated dissociation (EAD), that provides diagnostic fragment ions for the structural characterization of singly charged small molecules. Lipid mediators are derived from a limited number of precursor fatty acids. They are distinguished not necessarily by mass but by the location of their hydroxyl functional group(s), double bonds, and, potentially, carbon rings. Consequently, many of these compounds are isomeric at the precursor and the CID-based product ion levels; hence, the identification and quantitation of these compounds have relied heavily on careful chromatographic separation to achieve isomeric resolution. The ZenoTOF 7600 instrument, however, has a tunable electron beam to produce EAD-based fragments that can distinguish lipid mediator isomers.
OBJECTIVES:
The objective of this study was to quantify and fully characterize lipid mediators and other small-molecule metabolites using both CID- and EAD-based fragmentation with the SCIEX ZenoTOF 7600 system. In particular, the use of EAD to achieve specific structural identification of lipid molecular species in simple and complex matrices was investigated.
METHODS:
Rats were subjected to an ischemic stroke (MCAO model) according to an institutionally approved protocol. Plasma samples were taken after 24 hours and extracted using a solid phase extraction (SPE) column. Extracts were reconstituted in 100 µL MeOH and stored at -20oC until analysis.
Samples were injected on a ExionLC system using a Phenomenex Kinetex C18 column for chromatographic separation. Mobile phases were (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in 84:16 (v/v) acetonitrile/ methanol. Gradient conditions were used for a total run time of 21 min. The injection volume was 10 µL, column oven was set to 60oC and flow rate was 0.4 mL/min. Analysis was performed on the ZenoTOF 7600 system with OptiFlow Turbo V ion source using scheduled, high-resolution multiple reaction monitoring (MRMHR) scan mode. Samples were acquired using both CID and EAD fragmentation.
RESULTS:
The high sensitivity of the ZenoTOF 7600 was able to detect endogenous levels of the lipid mediators in the control and ischemic stroke sample sets. For example, the relative changes in the 12,13-EpOME concentrations resulting from ischemic stroke were observed. In addition, since full product ion spectra were simultaneously acquired, MS/MS spectral library matching was used to provide additional compound detection confirmation.
The MarkerView software was used to evaluate differences between the two sample sets. PCA analysis of the endogenous concentrations between healthy and ischemic rats showed significant differences in some lipid mediators due to ischemic stroke. The expense and difficulty in acquiring authentic standards for each lipid mediator prohibited absolute quantitation for each lipid mediator; however, using a single deuterated standard, as was done in these experiments, enabled relative quantitation between multiple sample sets. These data were sufficient to identify lipid mediators that changed in response to the treatment and can be used to direct targeted efforts with the appropriate primary reference and internal standards.
The EAD fragmentation mode of the ZenoTOF 7600 system was used to produce unique fragments that were used to distinguish between isomeric compounds. For example, the prostaglandins, PGE2 and PGD2, have the same precursor mass (m/z 397.2 Da) and coeluted during the chromatographic gradient. Using CID-formed fragments – m/z 189 Da for PGE2 and m/z 233 Da for PGD2 – the two compounds could not be fully distinguished. However, the EAD fragmentation mode produced several unique fragments, such as m/z 227.067 Da for PGE2 and m/z 215.066 Da for PGD2. The two compounds could be fully distinguished using the EAD-formed fragments.
CONCLUSIONS:
In summary, the ZenoTOF 7600 system was used to measure approximately 90 different lipid mediators in vivo with a sensitivity like that of a high-end triple quadrupole mass spectrometry system. An added benefit to high-resolution quantitative analysis is the ability to collect an accurate mass product ion spectrum that can be used for structural characterization and confirmation. In particular, the EAD fragmentation mode was used to distinguish between isomeric compounds.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
