Plasma-Droplet Reaction Systems: A Direct Mass Spectrometry Approach for Enhanced Characterization of Lipids at Multiple Isomer Levels

Abstract

Neutral triacylglyceride (TG) lipids are critical in cellular function, signaling, and energy storage. Multiple molecular pathways control TG structure via nonselective routes making them structurally complex and analytically challenging to characterize. The presence of C=C bond positional isomers exacerbates this challenge as complete structural elucidation is not possible by conventional tandem mass spectrometric methods such as collision-induced dissociation (CID), alone. Herein, we report a custom-made coaxial contained-electrospray ionization (ESI) emitter that allows the fusion of plasma discharge with charged microdroplets during electrospray (ES). Etched capillaries were incorporated into this contained-ES emitter, facilitating the generation of reactive oxygen species (ROS) at low (3 kV) ESI voltages and allowing stable ESI ion signal to be achieved at an unprecedented high (7 kV) spray voltage. The analytical utility of inducing plasma discharge during electrospray was investigated using online ionization of neutral TGs, in situ epoxidation of unsaturation sites, and C=C bond localization via conventional CID mass spectrometry. Collisional activation of the lipid epoxide generated during the online plasma-droplet fusion experiment resulted in a novel fragmentation pattern that showed a quadruplet of diagnostic ions for confident assignment of C=C bond positions and subsequent isomer differentiation. This phenomenon enabled the identification of a novel TG lipid, composed of conjugated linoleic acid, that is isomeric with two other TG lipids naturally found in extra virgin olive oil. To validate our findings, we analyzed various standards of TG lipids, including triolein, trilinolein, and trilinolenin, and isomeric mixtures in the positive-ion mode, each of which produced the expected quadruplet diagnostic fragment ions. Further validation was obtained by analyzing standards of free fatty acids expected from the hydrolysis of the TG lipids in the negative-ion mode, together with isomeric mixtures. The chemistry governing the gas-phase fragmentation of the lipid epoxides was carefully elucidated for each TG lipid analyzed. This comprehensive shotgun lipidomic approach has the potential to impact biomedical research since it can be accomplished on readily available mass spectrometers without the need for instrument modification.

Scheme 1. Schematic Illustration of Structural Information Derived from Positive-Ion Mode Analysis of TGs and Negative-Ion Mode Analysis of FAs via (A) Conventional ESI Providing Only Side Chain and Head Group Information, Respectively, upon CID, and (B) Contained-ESI Capable of Plasma-Droplet Fusion Where the Combination of ROS Reactions and CID Yield Multiple Level of Isomer Differentiation, Including Head Group, Side Chain, and C=C Positional Isomers

Figure 1

Diagram showing the coaxial contained-ESI emitter for facilitation of plasma-droplet fusion reactions where lipid solution is delivered via deactivated fused silica capillary (orange line) and ammonium acetate solution is sprayed from etched silica (blue line) for in situ generation of ROS. Droplets from the two coaxial spray converge into an outer etched silica capillary. This outlet is operated in two modes: (i) Type I mode in which the inner capillaries protrude from the outer, facilitating droplet-phase reaction on a faster (μs) lifetime and (ii) Type II mode in which the inner capillaries regress into the outer capillary, creating a cavity that facilitates extended reagent mixing in a thin film for subsequent reactions on seconds timescale. The red thunder symbol indicates plasma generation from the etched silica capillary, which interacts with charged droplets from the fused silica.

Figure 2

Comparison of conventional deactivated fused silica emitters with chemically etched emitters during: (A) Negative-ion mode MS analysis of oleic acid (MW 282 Da) from conventional deactivated fused silica emitter (orange) and chemically etched silica (blue). The corresponding depiction of the Type I emitter configuration used (insets) shows the effect of spray voltage on epoxide product formation at m/z 297, which is prominent in etched emitter operation. (B) TIC of oleic acid analyses using deactivated fused silica (orange) and etched silica (blue) emitters under identical experimental conditions showing signal stability of the etched emitter versus that of the conventional emitter at varying spray voltage. (C) Plot of ROS reaction yield showing the production of oleic acid oxidation product for deactivated fused silica and etched silica capillaries under identical experimental conditions showing ROS onset formation at 3 kV spray voltage and increased reaction yield with increased spray voltage for the etched silica emitter.

Scheme 2. Schematic Showing Ionization (NH₄⁺ Adduct), Epoxidation, and CID of (A) TG Lipid with a Single C=C Bond Side Chain to Give Four Diagnostic Ions Per Double Bond: Two from Dissociation of Intact Lipid, and Two from Cleavage of the Lyso Form of the Lipid, (B) TG with a Two Double-Bond Side Chain Demonstrating the Importance of Analyzing MS/MS for Both the Single (+O) and Double (+2O) Epoxidation Products for Complete C=C Position Assignments for both Double Bonds

Figure 3

Positive-ion mode MS analysis of TGs via plasma-droplet fusing reactions: (A) full mass spectrum of triolein, with ammonium adduct at m/z 903. Inset MS/MS shows CID of the single epoxide product at m/z 919 giving 9Z C=C bond diagnostic ions 1, 2, 3, and 4. (B) Reaction scheme illustrating how the diagnostic ions are related to the lyso and intact triolein after fragmentation of the epoxide ion at m/z 919; (C) full mass spectrum of triolein isomer with 18:1 (6Z) side chains with the inset MS/MS showing CID of the single epoxide product where diagnostic ion 5, 6, 7, and 8 are detected. (D) Reaction scheme illustrating the relationship between diagnostic ions derived from lyso and intact form of the triolein isomer; (E) full mass spectrum of trilinolein, with ammonium adduct at m/z 897 and the corresponding epoxide products at intervals of 16 Da. Inset: MS/MS showing CID of the double epoxide product at m/z 929 giving 9Z diagnostic ions (9)/(10) and (13)/(14), along with 12Z diagnostic ions (11)/(12) and (15)/(16). (F) Reaction scheme showing the formation of the double epoxide product at m/z 929 with subsequent production of four MS/MS diagnostic ions for the 9Z position (blue) and four diagnostic ions for the 12Z position (green).

Figure 4

Positive-ion mode MS analysis of isomeric standard TG and complex olive oil samples. (A) Full mass spectrum of a mixture of two standard TGs isomers, one containing oleic acid (18:1, 9Z) side chains and another containing 18:1(6Z) side chains. The epoxide lipids were both observed at m/z 919, with the inset tandem MS showing expected quadruplet diagnostic ions for the 9Z and the 6Z positions, as fully described in (B). (C) Full mass spectrum of EVOO under plasma-droplet fusing conditions. (D) MS/MS of the epoxide peak at m/z 919 in (C), where inset (i) shows zoomed-in mass range for the lyso-form diagnostic ions and inset (ii) shows zoomed-in mass range for the intact form revealing the diagnostic ions that correspond to the presence of three isomeric TG lipids: TG 18:1(9)/18:1(9)/18:1(9), TG 18:2(9,12)_18:1(9)_18:0, and TG 18:2(10,12)_18:1(9)_18:0. The structures of these three TG isomers detected in EVOO are provided in (E) as well as their corresponding diagnostic ions in the groups of (i), (ii), and (iii) for the three TG isomers.

Figure 5

Negative-ion mode plasma-droplet contained-ESI MS analysis of (A) full mass spectrum of an isomeric mixture of free fatty acid standards oleic acid (18:1, 9Z) and cis-vaccenic acid (18:1, 11Z), with the Inset MS/MS showing CID of the single epoxide product at m/z 297 offering diagnostic ions for both isomers. (B) Full mass spectrum of standard mixture of linoleic acid (18:2, 9Z/12Z) and its conjugated linoleic acid (9Z/11E) isomer. The inset shows CID MS/MS of the single epoxide produce at m/z 295, revealing expected diagnostic ions for both isomers. (C) Full mass spectrum of standard isomeric mixture of α-linolenic acid (18:3, 9Z/12Z/15Z) and γ-linolenic acid (18:3, 6Z/9Z/12Z) with inset MS/MS of the single epoxide showing diagnostic ions for both isomers. (D) MS/MS of m/z 297 derived from analysis of the complex EVOO sample, which shows known diagnostic ions for FA 18:1(9) and FA 18:1(11) FAs. (E) MS/MS of m/z 295 derived from analysis of the complex EVOO sample showing diagnostic ions that confirm the presence of 18:2 (9,12) and 18:2 (10,12) side chain C=C positional isomers. (F) Reaction scheme highlighting diagnostic ions for the newly discovered FA 18:2 (10,12) C=C positional isomer.