FERONIA mutation induces high levels of chloroplast-localized Arabidopsides which are involved in root growth.

The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT-ICR)-MS approach was used for initial screening of wild-type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non-wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole-time-of-flight (Q-TOF) MS by direct injection and LC-MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild-type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild-type and fer mutant leaves. More significantly, five micron high-spatial resolution MALDI-MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.

Sputter-Coated Metal Screening for Small Molecule Analysis and High-Spatial Resolution Imaging in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles are efficient matrices in laser desorption/ionization (LDI) mass spectrometry (MS), especially for the profiling or imaging of small molecules. Recently, solvent-free physical vapor desorption (PVD), or sputter coating, was adopted as a homogenous method to rapidly apply metal nanoparticles (NPs) in situ to samples prior to LDI MS or MS imaging analysis. However, there has been no systematic study comparing different metal targets for the analysis of a variety of small molecule metabolites. Here, we present a screening and optimization of various sputter-coated metals, including Ag, Au, Cu, Pt, Ni, and Ti, for LDI analysis of small molecules in both positive and negative ion modes. Optimized sputter coating is then applied to high-spatial resolution LDI mass spectrometry imaging (MSI) of maize root and seed cross-sections. Noble metals, Ag, Au, and Pt, are found to be much more efficient than transition metals and organic matrices for most small metabolites. Sputter-coated metals are efficient for neutral lipids, such as triacylglycerols and diacylglycerols, but are very inefficient for most phospholipids. Graphical Abstract ᅟ.

Nanoparticle microarray for high-throughput microbiome metabolomics using matrix-assisted laser desorption ionization mass spectrometry.

A high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI)-MS-based metabolomics platform was developed using a pre-fabricated microarray of nanoparticles and organic matrices. Selected organic matrices, inorganic nanoparticle (NP) suspensions, and sputter coated metal NPs, as well as various additives, were tested for metabolomics analysis of the turkey gut microbiome. Four NPs and one organic matrix were selected as the optimal matrix set: α-cyano-4-hydroycinnamic acid, Fe3O4 and Au NPs in positive ion mode with 10 mM sodium acetate, and Cu and Ag NPs in negative ion mode with no additive. Using this set of five matrices, over two thousand unique metabolite features were reproducibly detected across intestinal samples from turkeys fed a diet amended with therapeutic or sub-therapeutic antibiotics (200 g/ton or 50 g/ton bacitracin methylene disalicylate (BMD), respectively), or non-amended feed. Among the thousands of unique features, 56 of them were chemically identified using MALDI-MS/MS, with the help of in-parallel liquid chromatography (LC)-MS/MS analysis. Lastly, as a proof of concept application, this protocol was applied to 52 turkey cecal samples at three different time points from the antibiotic feed trial. Statistical analysis indicated variations in the metabolome of turkeys with different ages or treatments. Graphical abstract ᅟ.

High-Spatial Resolution Mass Spectrometry Imaging: Toward Single Cell Metabolomics in Plant Tissues.

Mass spectrometry imaging (MSI) is a powerful tool that has advanced our understanding of complex biological processes by enabling unprecedented details of metabolic biology to be uncovered. Through the use of high-spatial resolution MSI, metabolite localizations can be obtained with high precision. Here we describe our recent progress to enhance the spatial resolution of matrix-assisted laser desorption/ionization (MALDI) MSI from ∼50 µm with the commercial configuration to ∼5 µm. Additionally, we describe our efforts to develop a 'multiplex MSI' data acquisition method to allow more chemical information to be obtained on a single tissue in a single instrument run, and the development of new matrices to improve the ionization efficiency for a variety of small molecule metabolites. In combination, these contributions, along with the efforts of others, will bring MSI experiments closer to achieving metabolomic scale.

Overlapping MALDI-Mass Spectrometry Imaging for In-Parallel MS and MS/MS Data Acquisition without Sacrificing Spatial Resolution.

Metabolomics experiments require chemical identifications, often through MS/MS analysis. In mass spectrometry imaging (MSI), this necessitates running several serial tissue sections or using a multiplex data acquisition method. We have previously developed a multiplex MSI method to obtain MS and MS/MS data in a single experiment to acquire more chemical information in less data acquisition time. In this method, each raster step is composed of several spiral steps and each spiral step is used for a separate scan event (e.g., MS or MS/MS). One main limitation of this method is the loss of spatial resolution as the number of spiral steps increases, limiting its applicability for high-spatial resolution MSI. In this work, we demonstrate multiplex MS imaging is possible without sacrificing spatial resolution by the use of overlapping spiral steps, instead of spatially separated spiral steps as used in the previous work. Significant amounts of matrix and analytes are still left after multiple spectral acquisitions, especially with nanoparticle matrices, so that high quality MS and MS/MS data can be obtained on virtually the same tissue spot. This method was then applied to visualize metabolites and acquire their MS/MS spectra in maize leaf cross-sections at 10 µm spatial resolution. Graphical Abstract ᅟ.

Large Scale Nanoparticle Screening for Small Molecule Analysis in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles (NPs) have been suggested as efficient matrixes for small molecule profiling and imaging by laser-desorption ionization mass spectrometry (LDI-MS), but so far there has been no systematic study comparing different NPs in the analysis of various classes of small molecules. Here, we present a large scale screening of 13 NPs for the analysis of two dozen small metabolite molecules. Many NPs showed much higher LDI efficiency than organic matrixes in positive mode and some NPs showed comparable efficiencies for selected analytes in negative mode. Our results suggest that a thermally driven desorption process is a key factor for metal oxide NPs, but chemical interactions are also very important, especially for other NPs. The screening results provide a useful guideline for the selection of NPs in the LDI-MS analysis of small molecules.

Multi-matrix, dual polarity, tandem mass spectrometry imaging strategy applied to a germinated maize seed: toward mass spectrometry imaging of an untargeted metabolome.

Mass spectrometry imaging (MSI) provides high spatial resolution information that is unprecedented in traditional metabolomics analyses; however, the molecular coverage is often limited to a handful of compounds and is insufficient to understand overall metabolomic changes of a biological system. Here, we propose an MSI methodology to increase the diversity of chemical compounds that can be imaged and identified, in order to eventually perform untargeted metabolomic analysis using MSI. In this approach, we use the desorption/ionization bias of various matrixes for different metabolite classes along with dual polarities and a tandem MSI strategy. The use of multiple matrixes and dual polarities allows us to visualize various classes of compounds, while data-dependent MS/MS spectra acquired in the same MSI scans allow us to identify the compounds directly on the tissue. In a proof of concept application to a germinated corn seed, a total of 166 unique ions were determined to have high-quality MS/MS spectra, without counting structural isomers, of which 52 were identified as unique compounds. According to an estimation based on precursor MSI datasets, we expect over five hundred metabolites could be potentially identified and visualized once all experimental conditions are optimized and an MS/MS library is available. Lastly, metabolites involved in the glycolysis pathway and tricarboxylic acid cycle were imaged to demonstrate the potential of this technology to better understand metabolic biology.