2-Hydroxy-5-nitro-3-(trifluoromethyl)pyridine as a Novel Matrix for Enhanced MALDI Imaging of Tissue Metabolites.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a label-free imaging technique, determines the spatial distribution and relative abundance of versatile endogenous metabolites in tissues. Meanwhile, matrix selection is generally regarded as a pivotal step in MALDI tissue imaging. This study presents the first report of a novel MALDI matrix, 2-hydroxy-5-nitro-3-(trifluoromethyl)pyridine (HNTP), for the in situ detection and imaging of endogenous metabolites in rat liver and brain tissues by MALDI-MS in positive-ion mode. The HNTP matrix exhibits excellent characteristics, including strong ultraviolet absorption, µm-scale matrix crystals, high chemical stability, low background ion interference, and high metabolite ionization efficiency. Notably, the HNTP matrix also shows superior detection capabilities, successfully showing 185 detectable metabolites in rat liver tissue sections. This outperforms the commonly used matrices of 2,5-dihydroxybenzoic acid and 2-mercaptobenzothiazole, which detect 145 and 120 metabolites from the rat liver, respectively. Furthermore, a total of 152 metabolites are effectively detected and imaged in rat brain tissue using the HNTP matrix, and the spatial distribution of these compounds clearly shows the heterogeneity of the rat brain. The results demonstrate that HNTP is a new and powerful positive-ion mode matrix to enhance the analysis of metabolites in biological tissues by MALDI-MSI.

Enhancement of in situ detection and imaging of phytohormones in plant tissues by MALDI-MSI using 2,4-dihydroxy-5-nitrobenzoic acid as a novel matrix.

Phytohormones possess unique chemical structures, and their physiological effects are regulated through intricate interactions or crosstalk among multiple phytohormones. MALDI-MSI enables the simultaneous detection and imaging of multiple hormones. However, its application for tracing phytohormones is currently restricted by low abundance of hormone in plant and suboptimal matrix selection. 2,4-Dihydroxy-5-nitrobenzoic acid (DHNBA) was reported as a new MALDI matrix for the enhanced detection and imaging of multiple phytohormones in plant tissues. DHNBA demonstrates remarkable sensitivity improvement when compared to the commonly used matrix, 2,5-dihydroxybenzoic acid (DHB), in the detection of isoprenoid cytokinins (trans-zeatin (tZ), dihy-drozeatin (DHZ), meta-topolin (mT), and N6-(Δ2-isopentenyl) adenine (iP)), jasmonic acid (JA), abscisic acid (ABA), and 1-aminocyclo-propane-1-carboxylic acid (ACC) standards. The distinctive properties of DHNBA (i.e. robust UV absorption, uniform matrix deposition, negligible background interference, and high ionization efficiency of phytohormones) make it as an ideal matrix for enhanced detection and imaging of phytohormones, including tZ, DHZ, ABA, indole-3-acetic acid (IAA), and ACC, by MALDI-MSI in various plant tissues, for example germinating seeds, primary/lateral roots, and nodules. Employing DHNBA significantly enhances our capability to concurrently track complex phytohormone biosynthesis pathways while providing precise differentiation of the specific roles played by individual phytohormones within the same category. This will propel forward the comprehensive exploration of phytohormonal functions in plant science.

2,5-Dihydroxyterephthalic Acid: A Matrix for Improved Detection and Imaging of Amino Acids.

Amino acids (AAs), which are low-molecular-weight (low-MW) metabolites, serve as essential building blocks not only for protein synthesis but also for maintaining the nitrogen balance in living systems. In situ detection and imaging of AAs are crucial for understanding more complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free mass spectrometric imaging technique that enables the simultaneous detection and imaging of the spatial distribution and relative abundance of different endogenous/exogenous compounds in biological samples. The excellent efficiency of MALDI-MSI is attributed to the choice of the MALDI matrix. However, to the best of our knowledge, no matrix has been specifically developed for AAs. Herein, we report a MALDI matrix, 2,5-dihydroxyterephthalic acid (DHT), which can improve the detection and imaging of AAs in biological samples by MALDI-MS. Our results indicated that DHT exhibited strong ultraviolet-visible (UV-vis) absorption, uniform matrix deposition, and high vacuum stability. Moreover, the matrix-related ion signals produced from DHT were reduced by 50 and 71.8% at m/z < 500 compared to the commonly used matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), respectively, in their respective organic solvents. In terms of quantitative performance, arginine, glutamic acid, glutamine, and proline can be detected with limits of detection of 6, 4, 6, and 4 ng/mL, respectively, using the DHT as the matrix. Using DHT as the matrix, all 20 protein AAs were successfully detected in human serum by MALDI-MS, whereas only 7 and 10 AAs were detected when DHB and CHCA matrices were used, respectively. Furthermore, 20 protein AAs and taurine were successfully detected and imaged in a section of edible Crassostrea gigas (oyster) tissue for the first time. Our study demonstrates that using DHT as a matrix can improve the detection and imaging of AAs in biological samples by MALDI-MS.