On-tissue boronic acid derivatization for the analysis of vicinal diol metabolites in maize with MALDI-MS imaging

Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross-sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high-resolution accurate mass measurements. In addition, reaction interference and cross-reactivity with various other functional groups were systematically studied to verify data interpretation.


| INTRODUCTION
Mass spectrometry imaging (MSI) is an important analytical tool that provides spatial and chemical information of metabolites in tissue samples. 1,2 As MSI has become more prevalent, so has the desire to improve the spatial resolution, thus allowing for the localization of metabolites down to the cellular and subcellular level. 1,2 Matrix-assisted laser desorption/ionization (MALDI)-MSI is an attractive technique towards high spatial resolution imaging due to its soft ionization and small sampling size. [3][4][5][6] In MALDI-MSI, achieving high spatial resolution leads to a decrease in the sampling size per pixel. Due to small sampling size, low abundance compounds may not be detected, especially those with low ionization efficiency. This limitation can be resolved by performing on-tissue chemical derivatization to selectively enhance targeted classes of compounds.
On-tissue chemical derivatization reactions can increase the ionization efficiency of analytes by providing the analyte either a permanent charge or high proton affinity. [7][8][9] Derivatization reactions using Girard's reagent T, 2-picolylamine, and coniferyl aldehyde have been successfully applied to modify carbonyl, carboxylic acid, and primary amine functional groups on tissue, respectively. [7][8][9] Boronic acids have been used to derivatize sugars, brassinosteroids, and other vicinal diol metabolites to form boronic esters in solution. [10][11][12] Recently, this method has been adapted by Kaya et al. for MALDI-MSI using a custom-synthesized boronic acid, 4-(N-methyl)pyridinium boronic acid. In this study, the synthesized boronic acid was used to visualize catecholamines in adrenal tissues. 13 Here, we adopt a commercially available boronic acid with a tertiary amine, 4-(dimethylamino)phenylboronic acid (DBA), for in situ chemical modification MSI of vicinal diols and applied this strategy to explore the metabolite coverage in maize stems, roots, and leaves, determining as many possible features that were the result of this reaction. As a result, a reactivity screening had to be conducted to determine what side reactions occur, so false identification could be minimized.  ing reaction efficiency, three sets of replicates were analyzed for both derivatized and underivatized samples in positive ion mode using silver as a matrix. Acidic compounds were also analyzed in negative ion mode using DAN as a matrix.

| Maize tissue growth
B73 maize stems and roots were grown as described in Dueñas et al. 9 In brief, maize seeds were arranged along the top of a moist paper towel. The seeds were staggered with their embryos facing down, and DI water was used to wet the paper towel. Then, the paper towel was rolled tight enough to prevent the maize seeds from moving. The roll was then secured with tape and placed in a 1 L beaker half-filled with DI water. The beaker was placed in the dark, and the seeds were allowed to grow. Once the roots had grown to 10-14 cm in length (approximately 10 days), the tissue was harvested and collected 1 cm above and below the seed, for the stem and root, respectively.
For maize leaf imaging, maize seeds were planted in soil and grown in a climate-controlled green house as described in Dueñas et al. 9 In the greenhouse, 30% humidity was maintained under a diurnal cycle of 16 h of light at 27 C and 8 h of dark at 24 C. The sections of leaves were harvested 11 days after planting and collected at the midpoint of the third true leaf.
Following harvest, the plant tissue samples were flash-frozen in 10% (w/v) porcine gelatin solution in a cryo-mold and flash-frozen using liquid nitrogen. Tissue samples were then cryo-sectioned at 10 μm thickness at −20 C and collected using Cryo-Jane tape (Leica Biosystems, Wetzlar, Germany) as described in Korte et al. 2 Prior to derivatization and matrix application, the samples were vacuum dried while gradually warming to ambient temperature.

| Matrix application
A sputter coater (Ted Pella, Redding, CA, USA) was used to apply a  respectively, but in very low abundance (see Figure 1D). When comparing the signal of catechol before and after the derivatization reaction, the conversion percentage was 98% at the optimized condition and the signal improved about sixfold. Moreover, there is also a cluster of peaks around m/z 440.249 corresponding to the boroxine of DBA ( Figure 1C and Scheme 1C), three boronic acids forming a cyclic structure with the loss of three waters, which has been previously reported to be formed by heating boronic acid. 16,17 Boroxine synthesis may be favored when boronic acid is applied to sample surface under nitrogen curtain gas, even at temperatures just above room temperature. The boroxine signal could be reduced by using a lower concentration of boronic acid; however, a slight excess of boronic acid is used to ensure the maximal derivatization of diols.

| Investigation of side reactions of boronic acid
Due to using excess boronic acid in the reaction, double derivatization  Figure 3B. This type of side reactions with carboxylic acids is common as confirmed using a myristic acid standard, but the signal is very low as shown in Figure 4. To avoid side reactions with organic matrices, LDI-MS can be performed without matrix as suggested by Kaya et al. 13 or nonorganic matrices can be adopted such as silver sputtering which was used in this study. The ion signal increased by 20-fold when using silver as a matrix for the DBA-derivatized catechol compared to when no matrix was used.
To better understand the side reactions of DBA, a systematic study was performed to determine the reaction efficiency of selected standard compounds reacting with DBA (Table 1) Reaction efficiency % ð Þ= reacted amount initial amount Here   (Table S1).

| On-tissue derivation of boronic acid for MS imaging
Tentative assignments were made using the Maize Genetics and Genomics Database and the METLIN library using a mass tolerance of 10 ppm. 18,19 Features were assigned accounting for the potential of hydrogen loss (Scheme 1A), molecular radical ion, and protonated ion, as well as possible adduct formation with a sodium ion, potassium ion, or silver ion. Using this screening method, 10 features were tentatively assigned by comparing the exact masses of known maize metabolites in the Maize Genetics and Genomics Database. An additional 23 peaks were tentatively assigned by exact mass matching using the METLIN library and having a known presence in plant tissues, although not necessarily in maize. These 33 tentative identifications are listed in Table 2, and their MS images are shown in  Table S2 and shown in Figures S2B, S3B, and S4B for leaf, root, and stem crosssections, respectively.
The potential side reaction with carboxylic acids was also explored among the 84 unique features in the derivatized tissue sections by subtracting the corresponding mass change and comparing with Maize Genetics and Genomics Database and METLIN library.
There were 34 features that could be assigned as a carboxylic acid metabolite based on exact mass and the compound having a known presence in plant tissue. Since the reaction efficiency with fatty acids is low (e.g., 3% for myristic acid), many of 34 assignment might not be real. Considering the fact that the ion signal of unreacted fatty acids is much higher in negative mode than that of the derivatized in positive mode (e.g., 50 times for myristic acid), maize tissues were analyzed in the negative mode to test how many of them are consistent among the 34 potential assignments as carboxylic acids. Among those, only five ( KDO is a metabolite produced by both bacteria and higher-level plants. 20 In plants, KDO is observed predominantly in young leaves and is present in the epidermis as shown in Figure 5. This metabolite is a precursor to lipid A, a part of LPS, which has a role as an inducer of systematic acquired resistance. In a previous study, LPS was determined to be localized to the epidermis of Arabidopsis leaves, matching the localization that we observed with KDO in maize. 21 Many glycosides were also tentatively assigned including arbutin, vanilloloside dihydroxyphenyl-galloyl-glucopyranoside, and hydroxyinol[glucosyl-glucoside]. The peak at m/z 401.164 was assigned as arbutin, which was present in the epidermis of the leaf as shown in Figure 5. Arbutin is a glycosylated hydroquinone, which has been reported to be present in leaves as a defense against drought. 22 The localization of arbutin to the epidermis supports this finding. The F I G U R E 5 Optical images and mass spectrometry (MS) images for maize leaf vicinal diol metabolites. The same intensity scale is used to produce false-color images between the derivatized and underivatized images for each m/zion cuticle wax of leaves protects plants from losing water during drought and other instances of stress. Since arbutin serves a protective role against drought, its localization to the epidermis is expected. The derivatized peak at m/z 218.098 is most likely glyceraldehyde (diol) or lactate (hydroxyl and carboxylic acid in vicinal position) and observed in both root and stem tissues (Figures 6   and 7). In both these tissues, this metabolite is distributed throughout the pith, pericycle, endodermis, cortex, and epidermis but is absent from the xylem (tissue anatomy in Figure 6). Unfortunately, glyceraldehyde and lactate cannot be distinguished with accurate mass alone as they all have the same molecular formulae, C 3 H 6 O 3 .
In addition, performing MS/MS on these derivatized compounds mostly results in fragments that correspond to the derivatization agent, DBA; thus, we were not able to confidently confirm compound identifications. Nevertheless, it is very likely both are present in plant tissues.
F I G U R E 6 Optical images and mass spectrometry (MS) images for maize root vicinal diol metabolites. The same intensity scale is used to produce false-color images between the derivatized and underivatized images for each m/z ion. Underivatized MS images correspond to the silver ion adduct, unless denoted with an # or * which are sodiated and protonated, respectively

| CONCLUSION
In this study, in situ derivatization of vicinal diol compounds with a tert-amine containing boronic acid was investigated to improve the ion signals of vicinal diols for MALDI-MSI. Potential issues of this reaction were investigated including reaction suppression by sodium ions and side reactions with organic matrices. A systematic study was performed for the reaction with various chemical functional groups.
There was minimum side reaction for compounds with a carboxylic acid (conversion percentage, 3%-4%), but significant side reaction was observed for aromatic compounds with carboxylic and hydroxyl group in o-or m-position (50%-80%). By identifying these side reactions, more confident tentative assignments could be made. In addition, a significant increase in ion signal was demonstrated for vicinal diols and the efficient detection of new metabolite features was made possible from the 10 B/ 11 B isotope pattern. The optimized reaction condition was successfully applied to maize tissues of leaf, root, and stem, detecting dozens of metabolite features and their localizations, which was not possible without derivatization.