Mass Spectrometry Imaging of Lipids with Isomer Resolution Using High-Pressure Ozone-Induced Dissociation

Abstract

Mass spectrometry imaging (MSI) of lipids within tissues has significant potential for both biomolecular discovery and histopathological applications. Conventional MSI technologies are, however, challenged by the prevalence of phospholipid regioisomers that differ only in the location(s) of carbon-carbon double bonds and/or the relative position of fatty acyl attachment to the glycerol backbone (i.e., sn position). The inability to resolve isomeric lipids within imaging experiments masks underlying complexity, resulting in a critical loss of metabolic information. Herein, ozone-induced dissociation (OzID) is implemented on a mobility-enabled quadrupole time-of-flight (Q-TOF) mass spectrometer capable of matrix-assisted laser desorption/ionization (MALDI). Exploiting the ion mobility region in the Q-TOF, high number densities of ozone were accessed, leading to ∼1000-fold enhancement in the abundance of OzID product ions compared to earlier MALDI-OzID implementations. Translation of this uplift into imaging resulted in a 50-fold improvement in acquisition rate, facilitating large-area mapping with resolution of phospholipid isomers. Mapping isomer distributions across rat brain sections revealed distinct distributions of lipid isomer populations with region-specific associations of isomers differing in double bond and sn positions. Moreover, product ions arising from sequential ozone- and collision-induced dissociation enabled double bond assignments in unsaturated fatty acyl chains esterified at the noncanonical sn-1 position.

Figure 1

MALDI-OzID of monounsaturated (A–C) [PC 34:1 + Na]⁺ and (D–F) [PC 36:1 + Na]⁺, showing different isomer distributions in rat brain. MALDI-OzID spectrum of (A) [PC 34:1 + Na]⁺ and (D) [PC 36:1 + Na]⁺, where labeled product ions indicate the presence of n-7 and n-9 db isomers. Distribution of precursor ions (B) [PC 34:1 + Na]⁺ at m/z 782.57 and (E) [PC 36:1 + Na]⁺ at m/z 810.61 extracted from TOF-only scans. Fractional distribution images of (C) PC 34:1n-7 and (F) PC 36:1n-7 obtained from MALDI-OzID, showing relative changes in n-7 isomers throughout the rat brain. In (B, C) and (E, F), H&E staining of the same tissue after the MSI experiments is shown on the left. Note that MALDI-OzID and MALDI-TOF images are acquired from different brain tissue sections from the same animal. Explanation of additional ions present in (A) is provided as Supporting Information Figure S6. A technical replicate of these experiments with MS1 and OzID measurements acquired on consecutive sections is provided in Figure S8.

Figure 2

MALDI-OzID of polyunsaturated PC lipids showing the distribution of the polyunsaturated phospholipid and an interfering lipid throughout the rat brain, showing different distributions in white/gray matter. (A) MALDI-OzID spectrum of [PC 38:4 + Na]⁺, revealing the presence of an omega-6 fatty acid. In addition, n-9 OzID fragments from an interference are visible in the spectrum, labeled as n-9. (B) Distribution images of the omega-6 and (C) the n-9 OzID fragments. (D) MALDI-OzID spectrum of [PC 38:6 + Na]⁺, revealing the presence of an omega-3 fatty acid. In addition, n-9 OzID fragments from an interference are visible in the spectrum, labeled as n-9. (E) Distribution images of the omega-3 and (F) the n-9 OzID fragments. For these images, 99th quantile hotspot removal was applied to the non-normalized data.

Figure 3

MALDI-COzID of (A–D) [PC 34:1 + Na]⁺ and (E–J) [PC 36:1 + Na]⁺ in rat brain obtained by preactivating the ions with CID (40 V) in the trap region prior to ozonolysis in the ion mobility cell. (A) COzID mass spectrum of [PC 34:1 + Na]⁺ showing diagnostic product ion pairs indicating the presence of db-positional (PC 34:1n-7 and PC 34:1n-9) and sn-positional (PC 16:0/18:1 and PC 18:1/16:0) isomers. Additional product ions observed in the spectrum are consistent with CID/OzID² processes, which are regiospecific for the n-7 and n-9 db positions of the sn-1 18:1. (B–D) FDIs of the different isomers present, showing the intensity of signals specific for (B) PC 16:0/18:1, (C) PC 34:1n-7, and (D) [PC 18:1(n-7)/16:0 + Na]⁺ isomers. (E) COzID spectrum of [PC 36:1 + Na]⁺ showing diagnostic product ion pairs indicating the presence of db-positional (PC 36:1n-7 and PC 36:1n-9) and sn-positional (PC 18:0/18:1 and PC 18:1/18:0, PC 16:0/20:1 and PC 20:1/16:0) isomers. Additional product ions observed in the spectrum are consistent with CID/OzID² processes, which are regiospecific for the n-7 and n-9 db positions of the sn-1 18:1 and sn-1 20:1. (F–J) FDIs of the different isomers present, showing the intensity of (F) PC 18:0/18:1, (G) PC 16:0/20:1, (H) PC 36:1(n-7) db position, (I) [PC 18:1(n-7)/18:0 + Na]⁺, and (J) [PC 20:1(n-7)/16:0 + Na]⁺ isomers. For the FDIs, an H&E staining of a consecutive section is shown on the left.