Ozone-induced dissociation on a modified tandem linear ion-trap: Observations of different reactivity for isomeric lipids

Abstract

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation. In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell. This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca. 30-fold). These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s: significantly enhancing the utility of OzID in high-throughput lipidomic protocols. The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry. For cis-and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry. Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions. This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution. (J Am Soc Mass Spectrom 2010, 21, 1989 . There are a wide range of lipid subclasses with different biochemical roles, including glycerophospholipids (GPLs) that act as primary building blocks of membranes and precursors for intracellular signaling molecules; fatty acids (FAs) and triacylglycerols (TAGs) that are the major source of energy in plants and animals; and sterols that modulate membrane stability and act as biochemical messengers [2]. The specific functions of lipid classes, and indeed individual lipids, are related to their chemical and physical properties that in turn depend on specific molecular features [3]. As such, even small changes in molecular structure can affect the role of a lipid within a living organism. Recent research has indicated that within living organisms, different lipid isomers play different, and in some cases contrasting, metabolic roles. For example, one study focusing on the effect of conjugated linoleic acid isomers on development of atherosclerosis in ApoE knockout mice revealed that while one isomer (10E,12Z-18:2) had a profound atherogenic effect, an alternate isomer (9Z, 11E-18:2) was anti-atherogenic [4]. While differences in molecular structure arising from double-bond position and/or stereoisomerism (vide infra) can be cha...

Ozone-Induced Dissociation of Conjugated Lipids Reveals Significant Reaction Rate Enhancements and Characteristic Odd-Electron Product Ions

“…While CACI-MS/MS relies on an in-source chemical modification of lipid molecules prior to any mass selection, OzID begins with the conventional electrospray ionization (ESI) of lipids that are then mass-selected before undergoing ion-molecule reactions with ozone. This technique has been used to identify isomeric lipids differing in double-bond position from complex biological extracts, including olive oil [30], human lens [31], and cow brain [32]. Here, we demonstrate, for the first time, the effectiveness of OzID analysis on conjugated lipids and report a remarkable enhancement in the rate of the ion-molecule reaction in the presence of this bonding motif.…”

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

“…OzID product ions can be predicted and thus double bond positions can be confidently assigned without reference to standard compounds [29]. This, combined with the fact that the analysis is undertaken on mass-selected ions without the need for prior fractionation and can be achieved with low sample volumes [30] suggests that it is ideally suited to the structural interrogation of lipids involved in protein binding.…”

“…We have recently noted that ionized phospholipids bearing a trans double bond react with ozone at a greater rate than the corresponding cis-variants. This gives rise to greater peak intensities in the OzID spectra of trans versus cis lipids under the same experimental conditions [30]. While this has the advantage of allowing the analysis of a massselected and intact phospholipid, the characterization of the stereochemistry of an unknown lipid would require careful comparison to related standards, of which very few are readily available.…”

Rapid Identification and Quantification of Linear Olefin Isomers by Online Ozonolysis-Single Photon Ionization Time-of-Flight Mass Spectrometry

“…Target ion CH 2 [20][21][22][23][24][25]. Structurally characteristic fragments, including aldehydes and Criegee ions, occurred in the mass spectra recorded in the course of ozone electrospray ionization (OzESI) [21,24] and in ozone-induced dissociation (OzID) [23,25], which allowed assignment of double bond positions._ENREF_27 Both of OzESI and OzID simplified the complicated sample manipulation process needed prior to MS analysis so that the online location of double bond positions could be done more efficiently. Besides, OzID can be applied to the analysis of complex mixtures compared with OzESI, but it requires specialized equipment and instrumental modification.…”