A Cooperative Matrix for Enhanced MALDI MSI of Metabolic Alterations Associated with Oxidative Stress in Spinal Cord Injury

Abstract Oxidative stress following spinal cord injury (SCI) triggers diverse metabolic alterations, including those in the acrolein (ACR)-associated pathway, which involves reactive carbonyl species (RCSs), polyamines, amino acids, fatty acids, and lipids. Therefore, comprehensive mapping of these metabolites is critical for understanding disease progression of SCI. Although matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is widely used to map endogenous compounds, the simultaneous broad-spectrum detection of multiple analyte classes within the oxidative stress network remains challenging. Thus, we developed a cooperative matrix (co-matrix) composed of 2-hydrazinoquinoline (2-HQ) and α-cyano-4-hydroxycinnamic acid (CHCA) for the MALDI MSI of rat spinal cord tissue after SCI. In this system, ion-pair formed between 2-HQ and CHCA achieved crystallization uniformity and spot-to-spot reproducibility, while 2-HQ selectively derivatizes RCSs in situ. Density functional theory calculations further revealed that the ionicity of the co-matrix enhances analyte ionization. Using this co-matrix, we imaged 41 distinct metabolites in rat SCI tissue sections with high signal-to-noise ratios, including two carbonyl compounds associated with ACR-related oxidative stress pathways. In comparison, CHCA alone allowed imaging of only 15 metabolites (predominantly lipids), while 2-HQ alone detected one. Temporal heatmap analysis of nine metabolites within the ACR-associated pathway revealed coordinated dynamics between ACR and glutathione, both rebounding at day 7 (one-way ANOVA, p < 0.01). Overall, this study provides an efficient strategy for the comprehensive imaging analysis of multiple metabolite classes in complex injured biological systems.