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Multimodal mass spectrometry imaging (MSI) is a critical technique used for deeply investigating biological systems by combining multiple MSI platforms in order to gain the maximum molecular information about a sample that would otherwise be limited by a single analytical technique. The aim of this work was to create a multimodal MSI approach that measures metabolomic and proteomic data from a single biological organ by combining infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for metabolomic MSI and nanodroplet processing in one pot for trace samples (nanoPOTS) LC-MS/MS for spatially resolved proteome profiling. Adjacent tissue sections of rat brain were analyzed by each platform, and each data set was individually analyzed using previously optimized workflows. IR-MALDESI data sets were annotated by accurate mass and spectral accuracy using HMDB, METLIN, and LipidMaps databases, while nanoPOTS-LC-MS/MS data sets were searched against the rat proteome using the Sequest HT algorithm and filtered with a 1% FDR. The combined data revealed complementary molecular profiles distinguishing the corpus callosum against other sampled regions of the brain. A multiomic pathway integration showed a strong correlation between the two data sets when comparing average abundances of metabolites and corresponding enzymes in each brain region. This work demonstrates the first steps in the creation of a multimodal MSI technique that combines two highly sensitive and complementary imaging platforms. Raw data files are available in METASPACE (https://metaspace2020.eu/project/pace-2021) and MassIVE (identifier: MSV000088211).

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is a hybrid mass spectrometry ionization source that combines the benefits of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) making it a great analytical tool for high-throughput screening (HTS) analyses. IR-MALDESI is coupled to an Orbitrap Exploris 240 mass spectrometer that utilizes a bent quadrupole (C-trap) to inject accumulated ions into the high-field Orbitrap mass analyzer. Here, we present a study on the optimized C-trap timing for HTS analyses by IR-MALDESI mass spectrometry. The timing between initial ion generation and the C-trap opening time was optimized to reduce unnecessary ambient ion accumulation in the mass spectrometer. The time in which the C-trap was held open, the ion accumulation time, was further optimized to maximize the accumulation of analyte ions generated using IR-MALDESI. The resulting C-trap opening scheme benefits small-molecule HTS analyses by IR-MALDESI by maximizing target ion abundances, minimizing ambient ion abundances, and minimizing the total analysis time per sample. The proposed C-trap timing scheme for HTS does not translate to large molecules; a NIST monoclonal antibody standard reference material was analyzed to demonstrate that larger analytes require longer ion accumulation times and that IR-MALDESI can measure intact antibodies in their native state.

N -Linked glycans are structurally diverse polysaccharides that represent significant biological relevance due to their involvement in disease progression and cancer. Due to their complex nature, N -linked glycans pose many analytical challenges requiring the continued development of analytical technologies. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid ionization technique commonly used for mass spectrometry imaging (MSI) applications. Previous work demonstrated IR-MALDESI to significantly preserve sialic acid containing N -linked glycans that otherwise require chemical derivatization prior to detection. Here, we demonstrate the first analysis of N -linked glycans in situ by IR-MALDESI MSI. A formalin-fixed paraffin-embedded human prostate tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N -linked glycans. Fifty-three N -linked glycans were confidently identified in the prostate sample where more than 60% contained sialic acid residues. This work demonstrates the first steps in N -linked glycan imaging of biological tissues by IR-MALDESI MSI. Raw data files are available in MassIVE (identifier: MSV000088414).

Per- and polyfluoroalkyl substances (PFAS) are used extensively in commercial products. Their unusual solubility properties make them an ideal class of compounds for various applications. However, these same properties have led to significant contamination and bioaccumulation given their persistence in the environment. Development of analytical techniques to detect and quantify these compounds must take into account the potential for these properties to perturb these measurements, specifically the potential to bias the electrospray ionization (ESI) process. Direct injection ESI analysis of 23 different PFAS species revealed that hydrophobicity and PFAS class can predict the ESI overall response factors. In this study, a method for predicting the behavior of individual PFAS compounds, including relative retention order in chromatography, is presented which is simply based on the number of fluorine atoms in the molecule as well as the class of the compound (e.g., perfluroalkylcarboxylic acids) vs. computational estimations (e.g., non-polar surface area and logP)., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Glycosylation is a ubiquitous co- and post-translational modification involved in the sorting, folding, and trafficking of proteins in biological systems; in humans, >50% of gene products are glycosylated with the cellular machinery of glycosylation compromising ~2% of the genome. Perturbations in glycosylation have been implicated in a variety of diseases including neurodegenerative diseases and certain types of cancer. However, understanding the relationship between a glycan and its biological role is often difficult due to the numerous glycan isomers that exist. To address this challenge, nanoflow liquid chromatography, ion mobility spectrometry, and mass spectrometry (nLC-IMS-MS) were combined with the Individuality Normalization when Labeling with the Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy to study a series of glycan standards and those enzymatically released from the glycoproteins horseradish peroxidase, fetuin, and pooled human plasma. The combination of IMS and the natural (NAT) and stable-isotope label (SIL) in the INLIGHT™ strategy provided additional confidence for each glycan identification due to the mobility aligned NAT- and SIL-labeled glycans and further capabilities for isomer examinations. Additionally, molecular trend lines based on the IMS and MS dimensions were investigated for the INLIGHT™ derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Mass spectrometry imaging is a powerful tool to analyze a large number of metabolites with their spatial coordinates collected throughout the sample. However, the significant differences in ionization efficiency pose a big challenge to metabolomic mass spectrometry imaging. To solve the challenge and obtain a complete data profile, researchers typically perform experiments in both positive and negative ionization modes, which is time-consuming. In this work, we evaluated the use of the dicationic reagent, 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride (abbreviated to [C 5 (bpyr) 2 ]F 2 ) to detect a broad range of metabolites in the positive ionization mode by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI). [C 5 (bpyr) 2 ]F 2 at 10 µM was doped in 50% MeOH/H 2 O ( v / v ) electrospray solvent to form +1 charged adducted ions with anionic species (-1 charged) through post-electrospray ionization. This method was demonstrated with sectioned rat liver and hen ovary. A total of 73 deprotonated metabolites from rat liver tissue sections were successfully adducted with [C 5 (bpyr) 2 ] 2+ and putatively identified in the adducted positive ionization polarity, along with 164 positively charged metabolite ions commonly seen in positive ionization mode, which resulted in 44% increased molecular coverage. In addition, we were able to generate images of hen ovary sections showing their morphological features. Following-up tandem mass spectrometry (MS/MS) indicated that this dicationic reagent [C 5 (bpyr) 2 ] 2+ could form ionic bonds with the headgroup of glycerophospholipid ions. The addition of the dicationic reagent [C 5 (bpyr) 2 ] 2+ in the electrospray solvent provides a rapid and effective way to enhance the detection of metabolites in positive ionization mode.

Parkinson's disease (PD) is a neurodegenerative disorder commonly treated with levodopa (L-DOPA), which eventually induces abnormal involuntary movements (AIMs). The neurochemical contributors to these dyskinesias are unknown; however, several lines of evidence indicate an interplay of dopamine (DA) and oxidative stress. Here, DA and hydrogen peroxide (H 2 O 2 ) were simultaneously monitored at discrete recording sites in the dorsal striata of hemiparkinsonian rats using fast-scan cyclic voltammetry. Mass spectrometry imaging validated the lesions. Hemiparkinsonian rats exhibited classic L-DOPA-induced AIMs and rotations as well as increased DA and H 2 O 2 tone over saline controls after 1 week of treatment. By week 3, DA tone remained elevated beyond that of controls, but H 2 O 2 tone was largely normalized. At this time point, rapid chemical transients were time-locked with spontaneous bouts of rotation. Striatal H 2 O 2 rapidly increased with the initiation of contraversive rotational behaviors in lesioned L-DOPA animals, in both hemispheres. DA signals simultaneously decreased with rotation onset. The results support a role for these striatal neuromodulators in the adaptive changes that occur with L-DOPA treatment in PD and reveal a precise interplay between DA and H 2 O 2 in the initiation of involuntary locomotion., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa , two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa . The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Sun, Song, Chen, Shi, Yang, Liu, Tunlaya-Anukit, Liu, Loziuk, Williams, Muddiman, Lin, Sederoff, Wang and Chiang.)

Rationale: Radical cationization of endogenous hydrocarbons in cherry tomatoes was previously reported using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), a mass spectrometry imaging technique that operates at ambient conditions and requires no sample derivatization. Due to the surprising nature of this odd-electron ionization, subsequent experiments were performed on β-carotene to determine the amount of radical cationization across different sampling conditions., Methods: β-Carotene was analyzed across a variety of sample states using IR-MALDESI followed by Orbitrap mass spectrometric analysis: first, as a standard in ethanol in a well plate; second, as particulates on printer paper; and third, as particulates covered by an ice matrix. These techniques were also performed with a β-carotene standard either in solution with a reducing agent (ascorbic acid) or with ascorbic acid in the electrospray solution., Results: Tandem mass spectrometry confirmed the presence of the radical cation of β-carotene by comparing fragments against NIST and METLIN databases. It was always analyzed as a radical cation when sampled from solution, where ascorbic acid increased radical cation abundance when in solution with β-carotene. Mixed-mode ionization between radical cationization and proton adduction was observed from dried particulates using IR-MALDESI., Conclusions: There are several potential mechanisms for β-carotene radical cationization prior to IR-MALDESI analysis, with multiphoton ionization, thermal degradation, and/or reaction with oxygen appearing to be the most logical explanations. Furthermore, although not the primary cause, changing certain aspects of sample conditions can result in significant mixed-mode ionization with competing protonation., (© 2021 John Wiley & Sons Ltd.)

Introduction: Muscle invasive bladder cancer (MIBC) is an advanced stage of bladder cancer which poses a severe threat to life. Cancer development is usually accompanied by remarkable alterations in cell metabolism, and hence deep insights into MIBC at the metabolomic level can facilitate the understanding of the biochemical mechanisms involved in the cancer development and progression., Methods: In this proof-of-concept study, the optimal cutting temperature (OCT)-embedded MIBC samples were first washed with pure water to remove the polymer compounds which could cause severe signal suppression during mass spectrometry. Further, the tissue sections were analyzed by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI), providing an overview on the spatially resolved metabolomic profiles., Results: The MSI data enabled the discrimination between not only the cancerous and normal tissues, but also the subregions within a tissue section associated with different disease states. Using t-Distributed Stochastic Neighbor Embedding (t-SNE), the hyperdimensional MSI data was mapped into a two-dimensional space to visualize the spectral similarity, providing evidence that metabolomic alterations might have occurred outside the histopathological tumor border. Least absolute shrinkage and selection operator (LASSO) was further employed to classify sample pathology in a pixel-wise manner, yielding excellent prediction sensitivity and specificity up to 96% based on the statistically characteristic spectral features., Conclusion: The results demonstrate great promise of IR-MALDESI MSI to identify molecular changes derived from cancer and unveil tumor heterogeneity, which can potentially promote the discovery of clinically relevant biomarkers and allow for applications in precision medicine., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rationale: Unsaturated fatty acids (UFAs) play vital roles in regulating cellular functions. In-depth structural characterization of UFAs such as localizing carbon-carbon double bonds is fundamentally important but poses considerable challenges in mass spectrometry (MS) given that the most widely accessible ion activation method, low-energy collision-induced dissociation (CID), primarily generates uninformative fragments (e.g., neutral loss of CO 2 ) that are not suggestive of the double-bond positions., Methods: m-Chloroperoxybenzoic acid (mCPBA) was uniformly deposited onto the sample slides using a TM Sprayer, converting the carbon-carbon double bonds into epoxides under ambient conditions. The epoxidation product was ionized in situ by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS), and subsequently cleaved via CID, generating a diagnostic ion pair associated with the double-bond position. The reaction efficiency, sensitivity and relative quantification capability of the method were validated with five UFA standards dried on glass slides, and then this strategy was demonstrated on thin tissue sections of rat liver and human bladder., Results: The mCPBA reaction yielded conversion rates in the range of 44-60% in 10 min with high specificity and sensitivity. Further tandem mass spectrometry (MS/MS) of the mono-epoxidized products generated informative fragment ions specific to the double-bond positions, and relative quantification of positional isomers in binary mixtures was performed across a wide mole fraction from 0 to 1. An innovative spiral scan pattern was utilized during data acquisition, elucidating the major isomeric compositions of multiple UFAs from a tissue section in a single run., Conclusions: The on-tissue mCPBA epoxidation was implemented into an ambient MS imaging workflow to offer a rapid and simple way for in situ identification and relative quantification of double-bond positional isomers without the requirement for instrument modification. The method can be readily implemented on many other MS platforms to reveal the role of double-bond positional isomers in lipid biology and to discover potential biomarkers., (© 2021 John Wiley & Sons Ltd.)

Glycans are responsible for many biological activities; however, their structures are incredibly diverse and complex, often rendering the field of glycomics unsolvable by a single analytical technique. The development of multiple chemical derivatization strategies and bioinformatic software is responsible for some of the greatest analytical gains in the field of glycomics. The INLIGHT strategy is a chemical derivatization technique using hydrazide chemistry to derivatize the reducing end of N-linked glycans and incorporates either a natural (NAT, 12 C 6 ) or a stable-isotope label (SIL, 13 C 6 ) to carry out relative quantification. Here we present GlycoHunter, a user-friendly software created in MATLAB that enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pair is generated for relative quantification, including but not limited to, INLIGHT. GlycoHunter accepts the commonly used data file formats imzML and mzXML and effectively identifies all peak pairs associated with NAT- and SIL-labeled N-linked glycans using MS1 data. It also includes the ability to tailor the search parameters and export the results for further analysis using Skyline or Excel.

Three-dimensional (3D) mass spectrometry imaging (MSI) has become a growing frontier as it has the potential to provide a 3D representation of analytes in a label-free, untargeted, and chemically specific manner. The most common 3D MSI is accomplished by the reconstruction of 2D MSI from serial cryosections; however, this presents significant challenges in image alignment and registration. An alternative method would be to sequentially image a sample by consecutive ablation events to create a 3D image. In this study, we describe the use of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) in ablation-based 3D MSI for analyses of lipids within fresh frozen skin tissue. Depth resolution using different laser energy levels was explored with a confocal laser scanning microscope to establish the imaging parameters for skin. The lowest and highest laser energy level resulted in a depth resolution of 7 μm and 18 μm, respectively. A total of 594 lipids were putatively detected and detailed lipid profiles across different skin layers were revealed in a 56-layer 3D imaging experiment. Correlated with histological information, the skin structure was characterized with differential lipid distributions with a lateral resolution of 50 μm and a z resolution of 7 μm.

A vision-system driven platform, RastirX, has been constructed for mass spectrometry imaging (MSI) of arbitrary two-dimensional patterns. The user identifies a region of interest (ROI) by drawing on a live video image of the sample with the computer mouse. Motion commands are automatically generated to move the sample to acquire scan data for the pixels in the ROI. Synchronization of sample stage motion with laser firing and mass spectrometer (MS) scan acquisition is fully automated. RastirX saves a co-registered optical image and the scan location information needed to convert raw MS data into imzML format. Imaging an arbitrarily shaped ROI instead of the minimal enclosing rectangle reduces contamination from off-sample material and significantly reduces acquisition time.

To better understand cell-to-cell heterogeneity, advanced analytical tools are in a growing demand for elucidating chemical compositions of each cell within a population. However, the progress of single-cell chemical analysis has been restrained by the limitations of small cell volumes and minute cellular concentrations. Here, we present a rapid and sensitive method for investigating the lipid profiles of isolated single cells using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS). In this work, HeLa cells were dispersed onto a glass slide, and the cellular contents were ionized by IR-MALDESI and measured using a Q-Exactive HF-X mass spectrometer. Importantly, this approach does not require extraction and/or enrichment of analytes prior to MS analysis. Using this approach, 45 distinct lipid species, predominantly phospholipids, were detected and putatively annotated from the single HeLa cells. The proof-of-concept study demonstrates the feasibility and efficacy of IR-MALDESI-MS for rapid lipidomic profiling of single cells, which provides an important basis for future work on differentiation between normal and diseased cells at various developmental states, which can offer new insights into cellular metabolic pathways and pathological processes. Although not yet accomplished, we believe this approach can be readily used as an assessment tool to compare the number of identified species during source evolution and method optimization (intra-laboratory), and also disclose the complementary nature of different direct analytical approaches for the coverage of different types of endogenous analytes (inter-laboratory).Graphical abstract.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20-100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc) 1 (Gal) 2 (GlcNAc) 4 (Man) 3 (NeuAc) 1 and (Gal) 3 (GlcNAc) 5 (Man) 3 (NeuAc) 3 , was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

Background: Kava is an important neuroactive medicinal plant. While kava has a large global consumer footprint for its clinical and recreational use, factors related to its use lack standardization and the tissue-specific metabolite profile of its neuroactive constituents is not well understood., Results: Here we characterized the metabolomic profile and spatio-temporal characteristics of tissues from the roots and stems using cross-platform metabolomics and a 3D imaging approach. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry revealed the highest content of kavalactones in crown root peels and lateral roots. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging revealed a unique tissue-specific presence of each target kavalactone. X-ray micro-computed tomography analysis demonstrated that lateral roots have morphological characteristics suitable for synthesis of the highest content of kavalactones., Conclusions: These results provide mechanistic insights into the social and clinical practice of the use of only peeled roots by linking specific tissue characteristics to concentrations of neuroactive compounds., (© The Author(s) 2020. Published by Oxford University Press GigaScience.)

Magnaporthe oryzae ( M. oryzae ) is a pathogenic, filamentous fungus that is a primary cause of rice blast disease. The M. oryzae protein MGG_13065, SCF E3 ubiquitin ligase complex F-box protein, has been identified as playing a crucial role in the infection process, specifically, as part of the ubiquitin mediated proteolysis pathway. Proteins targeted by MGG_13065 E3 ligase are first phosphorylated and then ubiquitinated by E3 ligase. In this study, we used a label-free quantitative global proteomics technique to probe the role of ubiquitination and phosphorylation in the mechanism of how E3 ligase regulates change in virulence of M. oryzae . To do this, we compared the WT M. oryzae 70-15 strain with a gene knock out (E3 ligase KO) strain. After applying a ≥ 5 normalized spectral count cutoff, a total of 4432 unique proteins were identified comprised of 4360 and 4372 in the WT and E3 ligase KO samples, respectively. Eighty proteins drastically increased in abundance, while 65 proteins decreased in abundance in the E3 ligase KO strain. Proteins (59) were identified only in the WT strain; 13 of these proteins had both phosphorylation and ubiquitination post-translational modifications. Proteins (71) were revealed to be only in the E3 ligase KO strain; 23 of the proteins have both phosphorylation and ubiquitination post-translational modifications. Several of these proteins were associated with key biological processes. These data greatly assist in the selection of future genes for functional studies and enable mechanistic insight related to virulence.

We report the spatially resolved metabolic profiling of cherry tomatoes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), a mass spectrometry imaging (MSI) technique that operates at ambient conditions and requires no sample derivatization. Tomatoes were flash frozen, cryosectioned and imaged with adequate spatial resolution to distinguish between the major tissue structures of a tomato including the skin, mesocarp, endocarp, locular tissue, septum, placenta, seed and seed coating. Metabolites were imaged from 100-1200 m/z, enabling significant coverage of a diverse array of metabolites including amino acids and lipids along with the major secondary metabolite classes: terpenes, phenolics, glycosides, and alkaloids. During the metabolic profiling, we found endogenous carotenoid hydrocarbons, namely lycopene or its structural isomer β-carotene, ionized as radical cations. To our knowledge, this is the first demonstration of ionizing hydrocarbons in the MSI field.

By employing chip-based capillary zone electrophoresis coupled to high-resolution mass spectrometry, we profiled the plasma metabolome of 134 patients diagnosed with sporadic amyotrophic lateral sclerosis (ALS) (81 males and 53 females) and 118 individuals deemed healthy (49 males and 69 females). The most significant markers ( p < 0.01) were creatine, which was 49% elevated, and creatinine and methylhistidine, which were decreased by 20 and 24%, respectively, in ALS patients. The ratio of creatine versus creatinine increased 370 and 200% for male and female ALS patients, respectively. In addition, male ALS patients on an average had 5-13% lower amounts of seven essential amino acids, whereas females did not significantly differ from healthy controls. We developed two models using the metabolite abundances: (1) a classification model for the separation of ALS and healthy samples and (2) a classification model for the prediction of disease progression based on the ALS functional rating score. Utilizing a Monte Carlo cross-validation approach, a linear discriminant analysis model achieved a mean area under the receiver operating characteristic curve (AUC) of 0.85 (0.06) with a mean sensitivity of 80% (9%) and specificity of 78% (10%) for the separation of ALS and controls, respectively. A support vector machine classifier predicted progression categories with an AUC of 0.90 (0.06) with a mean sensitivity of 73% (10%) and a specificity of 86% (5%). Lastly, using a previously reported assay with a stable isotope-labeled ( 13 C 3 15 N 2 ) spike-in standard, we were unable to detect the exogenous neurotoxic metabolite, β-methylamino-l-alanine, in the free or protein-bound fraction of any of the 252 plasma samples.

Glycan analysis by mass spectrometry has rapidly progressed due to the interest in understanding the role of glycans in disease and tumor progression. Glycans are complex molecules that pose analytical challenges due to their isomeric compositions, labile character, and ionization preferences. This study sought to demonstrate infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) as a novel approach for the direct analysis of N -linked glycans. The glycoprotein bovine fetuin was chosen for this analysis as its glycome is well-characterized and heavily composed of sialylated glycans. Native N- linked glycans produced by enzymatic cleavage (via PNGase F) of bovine fetuin were analyzed directly by IR-MALDESI in both positive and negative ionization mode. In this study, we detected 12 N -linked glycans in negative mode and 4 N -linked glycans in positive mode, a significant increase in the amount of underivatized glycans detected by other ionization sources. Importantly, all N -linked glycans detected contained at least one sialic acid residue, which are known to be labile. This work represents a critical first step for N -linked glycan analysis by IR-MALDESI with future efforts directed at mass spectrometry imaging.

There is a growing need to understand the potential neurotoxicity of organophosphate flame retardants (OPFRs) and plasticizers because use and, consequently, human exposure, is rapidly expanding. We have previously shown in rats that developmental exposure to the commercial flame retardant mixture Firemaster 550 (FM 550), which contains OPFRs, results in sex-specific behavioral effects, and identified the placenta as a potential target of toxicity. The placenta is a critical coordinator of fetal growth and neurodevelopment, and a source of neurotransmitters for the developing brain. We have shown in rats and humans that flame retardants accumulate in placental tissue, and induce functional changes, including altered neurotransmitter production. Here, we sought to establish if OPFRs (triphenyl phosphate and a mixture of isopropylated triarylphosphate isomers) alter placental function and fetal forebrain development, with disruption of tryptophan metabolism as a primary pathway of interest. Wistar rat dams were orally exposed to OPFRs (0, 500, 1000, or 2000 μg/day) or a serotonin (5-HT) agonist 5-methoxytryptamine for 14 days during gestation and placenta and fetal forebrain tissues collected for analysis by transcriptomics and metabolomics. Relative abundance of genes responsible for the transport and synthesis of placental 5-HT were disrupted, and multiple neuroactive metabolites in the 5-HT and kynurenine metabolic pathways were upregulated. In addition, 5-HTergic projections were significantly longer in the fetal forebrains of exposed males. These findings suggest that OPFRs have the potential to impact the 5-HTergic system in the fetal forebrain by disrupting placental tryptophan metabolism., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chemical exposures can adversely impact fetal development. For many compounds, including common flame retardants, the mechanisms by which this occurs remain unclear, but emerging evidence suggests that disruption at the level of the placenta may play a role. Understanding how the placenta might be vulnerable to chemical exposures is challenging due to its complex structure. The primary objective of this study was to develop a method for detecting placental neurotransmitters and related metabolites without chemical derivatization so changes in the abundance and spatial distribution of neurotransmitters in rat placenta following chemical exposure could be determined using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging. Without chemical derivatization, 49 neurotransmitters and their related metabolites were putatively identified in untreated rat placenta sections using mass measurement accuracy and spectral accuracy. A few neurotransmitters were less abundant in placentas that were exposed to various flame retardants and were further investigated by KEGG metabolic pathway analysis. Many of these downregulated neurotransmitters shared the same enzyme responsible for metabolism, aromaticl-amino acid decarboxylase, suggesting a mechanistic role. These data constitute a new approach that could help identify novel mechanisms of toxicity in complex tissues. Graphical abstract.

The zebrafish ( Danio rerio ) is an ideal model for whole animal studies of lipid metabolism and lipid-related disease. In this work, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) was applied for direct visualization of lipid and metabolite distributions across various organs in whole-body zebrafish tissue sections. Detailed methods for overcoming the challenges of cryosectioning adult male zebrafish for MSI and complementary histological imaging are described. Representative two-dimensional ion maps demonstrated organ specific localization of lipid analytes allowing for visualization of areas of interest including the brain, liver, intestines, and skeletal muscle. A high resolving power mass spectrometer was utilized for accurate mass measurements, which permitted the use of open-source, web-based tools for MS 1 annotations including METASPACE and METLIN. Whole-body MSI with IR-MALDESI allowed for broad lipid coverage with high spatial resolution, illustrating the potential of this technique for studying lipid-related diseases using zebrafish as a model organism.

Rationale: Mass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin-related compounds using stable-isotope-labelled (SIL) indole-3-acetic acid (IAA) doped into agarose substrate., Methods: Wild-type Arabidopsis thaliana seedlings, sur2 and wei8 tar2 loss-of-function mutants, and YUC1 gain-of-function line were grown for 3 days in the dark in standard growth medium. SIL-IAA was doped into a 1% low-melting-point agarose gel and seedlings were gently laid on top for IR-MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post-acquisition by normalization of auxin-related compounds to SIL-IAA in the agarose. Amounts of auxin-related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest., Results: IAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR-MALDESI analyses. Indole-3-acetaldoxime was increased in sur2 mutants compared with wild-type and other mutants. Other auxin-related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants., Conclusions: Agarose was shown to be an appropriate sampling surface for IR-MALDESI mass spectrometry imaging of Arabidopsis seedlings. SIL-IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations., (© 2019 John Wiley & Sons, Ltd.)

Because of its high degree of selectivity and chemical resolution, mass spectrometry (MS) is rapidly becoming the analytical method of choice for high-throughput evaluations and clinical diagnostics. While advances in MS resolving power have increased by an order of magnitude over the past decade, advances in sample introduction are still needed for high-throughput screening applications where the time frame of chromatographic separation would limit the duty cycle. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization source that has been shown to be applicable for direct analyses and mass spectrometry imaging (MSI) of complex biological samples in a high-throughput manner. To increase a range of detectable features in IR-MALDESI experiments, we integrated the home-built ion source with a commercially available drift tube ion mobility spectrometer-mass spectrometer (IMS-MS) and analyzed small polar molecules, lipids, carbohydrates, and intact proteins. We also describe in detail how the pulsed ionization source was synchronized with IMS-MS.

Mass spectrometry imaging as a field has pushed its frontiers to three dimensions. Most three-dimensional mass spectrometry imaging (3D MSI) approaches require serial sectioning that results in a loss of biological information between analyzed slices and difficulty in reconstruction of 3D images. In this contribution, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) was demonstrated to be applicable for 3D MSI that does not require sectioning because IR laser ablates material on a micrometer scale. A commercially available over-the-counter pharmaceutical was used as a model to demonstrate the feasibility of IR-MALDESI for 3D MSI. Depth resolution (i.e., z-resolution) as a function of laser energy levels and density of ablated material was investigated. The best achievable depth resolution from a pill was 2.3 μm at 0.3 mJ/pulse. 2D and 3D MSI were performed on the tablet to show the distribution of pill-specific molecules. A 3D MSI analysis on a region of interest of 15 × 15 voxels across 50 layers was performed. Our results demonstrate that IR-MALDESI is feasible with 3D MSI on a pill, and future work will be focused on analyses of biological tissues.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25-30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60-75% ACN/40-25% H 2 O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45-55% ACN/55-45% H 2 O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging is a useful tool for identifying important meta-metabolomic features pertinent for enhancing our understanding of biological systems. Magnaporthe oryzae (M. oryzae) is a filamentous fungus that is the primary cause of rice blast disease. True to its name, M. oryzae primarily destroys rice crops and can also destroy other cereal crops as well. In a previous study, the F-box E3 ligase protein in M. oryzae was noted to be crucial for its growth and pathogenicity. In this study, we inoculated three separate sets of barley with wild-type M. oryzae, an F-box E3 ligase protein knock out of M. oryzae, and a control solution. Over the course of the infection (8 days), we imaged each treatment after development of an advanced polarity switching method, which allowed for the detection of low and high molecular weight compounds that ionize in positive or negative polarities. A set of features from initial experiments were chosen for another analysis using tandem mass spectrometry. Serotonin, a barley defense metabolite, was a compound identified in both positive and negative modes. Serotonin was putatively identified using MS1 data including carbon estimation and sulfur counting then confirmed based on tandem mass spectrometry fragmentation patterns. Metabolites in the melanin pathway, important for infection development of M. oryzae, were also identified using MS1 data but were unable to be confirmed with MS/MS due to their low abundances.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods., (© 2020 De Leoz et al.)

We report an effective strategy for direct analysis and two-dimensional (2D) matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) of mouse bones that underwent no chemical treatments prior to analysis. To unravel the chemistry in bones under near-physiological conditions, we cut a flash-frozen bone in half longitudinally, placed it in a mold facing flat side down, and poured Plaster of Paris on top of and around the bone. After Plaster of Paris had set, the bone with embedding material was removed from the mold, and placed on the IR-MALDESI imaging stage. Plaster of Paris acted as a fixture to keep every spot on the sample surface the same distance away from the laser focus. To demonstrate the feasibility of IR-MALDESI MSI for analyses of unmodified bones, we imaged bones derived from healthy and stroke-affected mice and generated ion heatmaps showing the spatial distribution of putatively annotated features., Competing Interests: CONFLICT OF INTEREST There are no conflicts to declare.

The internal energy deposited into analytes during the ionization process largely influences the extent of fragmentation, thus the appearance of the resulting mass spectrum. The internal energy distributions of a series of para-substituted benzyl pyridinium cations in liquid and solid-state generated by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) were measured using the survival yield method, of which results were subsequently compared with conventional electrospray ionization (ESI). The comparable mean internal energy values (e.g., 1.8-1.9 eV at a collision energy of 15 eV) and peak widths obtained with IR-MALDESI and ESI support that IR-MALDESI are essentially a soft ionization technique where analytes do not gain considerable internal energy during the laser-induced desorption process and/or lose energy during uptake into charged electrospray droplets. An unusual fragment ion, protonated pyridine, was only found for solid IR-MALDESI at relatively high collision energies, which is presumably resulted from direct ionization of the pre-charged analytes in form of salts. Analysis of tissue with an ice layer consistently yielded ion populations with higher internal energy than its counterpart without an ice layer, likely due to a substantially enhanced number of IR absorbers with ice. Further measurements with holo-myoglobin show that IR-MALDESI-MS retains the noncovalently bound heme-protein complexes under both native-like and denaturing conditions, while complete loss of the heme group occurred in denaturing ESI-MS, showing that the softness of IR-MALDESI is equivalent or superior to ESI for biomolecules.

This is a report on comprehensive characterization of cadmium (Cd)-exposed root proteomes in tomato using label-free quantitative proteomic approach. Two genotypes differing in Cd tolerance, Pusa Ruby (Cd-tolerant) and Calabash Rouge (Cd-sensitive), were exposed during 4 days to assess the Cd-induced effects on root proteome. The overall changes in both genotypes in terms of differentially accumulated proteins (DAPs) were mainly associated to cell wall, redox, and stress responses. The proteome of the sensitive genotype was more responsive to Cd excess, once it presented higher number of DAPs. Contrasting protein accumulation in cellular component was observed: Cd-sensitive enhanced intracellular components, while the Cd-tolerant increased proteins of extracellular and envelope regions. Protective and regulatory mechanisms were different between genotypes, once the tolerant showed alterations of various protein groups that lead to a more efficient system to cope with Cd challenge. These findings could shed some light on the molecular basis underlying the Cd stress response in tomato, providing fundamental insights for the development of Cd-safe cultivars. Graphical abstract.

Mass spectrometry imaging is a powerful tool widely used in biological, clinical, and forensic research, but its often poor repeatability limits its application for quantitative and large-scale analysis. A systematic evaluation of infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS) repeatability in absolute ion abundances during short- and long-term experiments was carried out on liver slices from the same rat with minimal biological variability to be expected. Results of median %RSDs ranging from 14 to 45, pooled %RMADs ranging from 11 to 33, and Pearson correlation coefficients ranging from 0.83 to 1.00 demonstrated an acceptable repeatability of IR-MALDESI-MS. Normalization is commonly applied for the purpose of accounting for analytical variability of spectra generated from different runs so as to reveal real biological differences. Nine data normalization strategies were performed on the rat liver data sets to examine their effects on reducing analytical variation, and further on a hen ovary data set containing more morphological features for the investigation of their impact on ion images. Results demonstrated that the majority of normalization approaches benefit data quality to some extent, and local normalization methods significantly outperform their global counterparts, resulting in a reduction of median %RSD up to 22. Local median normalization was found to be promisingly robust for both homogeneous and heterogeneous samples.

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3 + T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-μm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3 + T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography-mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3 + T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Drought is considered the major abiotic stress limiting crop productivity. This study seeks to identify proteins involved in the drought response in sugarcane stems submitted to drought stress. The integration of nuclei enrichment sample preparation with the shotgun proteomic approach results in great coverage of the sugarcane stem proteome with 5381 protein groups identified. A total of 1204 differentially accumulated proteins are detected in response to drought, among which 586 and 618 are increased and reduced in abundance, respectively. A total of 115 exclusive proteins are detected, being 41 exclusives of drought-stressed plants and 74 exclusives of control plants. In the control plants, most of these proteins are related to cell wall metabolism, indicating that drought affects negatively the cell wall metabolism. Also, 37 transcription factors (TFs) are identified, which are low abundant nuclear proteins and are differentially accumulated in response to drought stress. These TFs are associated to protein domains such as leucine-rich (bZIP), C2H2, NAC, C3H, LIM, Myb-related, heat shock factor (HSF) and auxin response factor (ARF). Increased abundance of chromatin remodeling and RNA processing proteins are also observed. It is suggested that these variations result from an imbalance of protein synthesis and degradation processes induced by drought., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Rationale: Identification of subregions under different pathological conditions on cancerous tissue is of great significance for understanding cancer progression and metastasis. Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS) can be potentially used for diagnostic purposes since it can monitor spatial distribution and abundance of metabolites and lipids in biological tissues. However, the large size and high dimensionality of hyperspectral data make analysis and interpretation challenging. To overcome these barriers, multivariate methods were applied to IR-MALDESI data for the first time, aiming at efficiently resolving mass spectral images, from which these results were then used to identify normal regions within cancerous tissue., Methods: Molecular profiles of healthy and cancerous hen ovary tissues were generated by IR-MALDESI-MS. Principal component analysis (PCA) combined with color-coding built a single tissue image which summarizes the high-dimensional data features. Pixels with similar color indicated similar composition. PCA results from healthy tissue were further used to test each pixel in cancerous tissue to determine if it is healthy. Multivariate curve resolution-alternating least squares (MCR-ALS) was used to obtain major spatial features existing in ovary tissues, and group molecules with the same distribution patterns simultaneously., Results: PCA as the predominating dimensionality reduction approach captured over 90% spectral variances by the first three PCs. The PCA images show the cancerous tissue is more chemically heterogeneous than healthy tissue, where at least four regions with different m/z profiles can be differentiated. PCA modeling assigns top regions of cancerous tissue as healthy-like. MCR-ALS extracted three and four major compounds from healthy and cancerous tissue, respectively. Evaluating similarities of resolved spectra uncovered the chemical components that were distinct in some regions on cancerous tissue, serving as a supplementary way to differentiate healthy and cancerous regions., Conclusions: Two unsupervised chemometric methods including PCA and MCR-ALS were applied for resolving and visualizing IR-MALDESI-MS data acquired from hen ovary tissues, improving the interpretation of mass spectrometry imaging results. Then possible normal regions were differentiated from cancerous tissue sections. No prior knowledge is required using either chemometric method, so our approach is readily suitable for unstained tissue samples, which allows one to reveal the molecular events happening during disease progression., (© 2018 John Wiley & Sons, Ltd.)

Bioactive peptides have been identified in lactic acid bacteria fermented foods including cultured milk, sourdough, and cured meats; however, their presence has not been investigated in fermented vegetables. In this study, infrared, matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry (MS) was employed to identify bioactive peptides in fermented cucumber. Natural and starter culture fermented cucumbers were prepared in triplicate in sodium chloride brines and compared to acidified cucumbers. Putative matches of known food-derived bioactive peptides were identified by direct analysis using IR-MALDESI-MS. Peptides were confirmed by IR-MALDESI MS/MS and quantified by LC-MS/MS. Three angiotensin converting enzyme (ACE) inhibitory peptides, IPP (0.42-0.49 mg/kg), LPP (0.30-0.33 mg/kg), and VPP (0.32-0.35 mg/kg) were formed in fermented cucumbers. A fourth ACE inhibitory peptide, KP (0.93-1.5 mg/kg), was enhanced 3-5 fold in fermented cucumbers compared with acidified cucumbers. This work demonstrates that lactic acid bacteria fermentation can enhance bioactive peptide content in vegetables., (Published by Elsevier Ltd.)

There is a pressing need to develop tools for assessing possible neurotoxicity, particularly for chemicals where the mode of action is poorly understood. Tetrabromobisphenol A (TBBPA), a highly abundant brominated flame retardant, has lately been targeted for neurotoxicity analysis by concerned public health entities in the EU and USA because it is a suspected thyroid disruptor and neurotoxicant. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used for the analysis of neurotransmitters in the brains of rats exposed to TBBPA in gestation and lactation through their mothers. Three neurotransmitters of interest were studied in three selected regions of the brain: caudate putamen, substantia nigra (SN), and dorsal raphe. Stable isotope labeled (SIL) standards were used as internal standards and a means to achieve relative quantification. This study serves as a demonstration of a new application of IR-MALDESI, namely that neurotransmitter distributions can be confidently and rapidly imaged without derivatization.

Analyzing mass spectrometry imaging data can be laborious and time consuming, and as the size and complexity of datasets grow, so does the need for robust automated processing methods. We here present a method for comprehensive, semi-targeted discovery of molecular distributions of interest from mass spectrometry imaging data, using widely available image similarity scoring algorithms to rank images by spatial correlation. A fast and powerful batch search method using a MATLAB implementation of structural similarity (SSIM) index scoring with a pre-selected reference distribution is demonstrated for two sample imaging datasets, a plant metabolite study using Artemisia annua leaf, and a drug distribution study using maraviroc-dosed macaque tissue. Graphical Abstract ᅟ.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

We describe a set of new tools for the detection and quantification of β-N-methylamino-L-alanine (BMAA) which includes a novel stable isotope-labeled BMAA standard ( 13 C 3 , 15 N 2 ) and a chip-based capillary electrophoresis mass spectrometry platform for separation and detection. Baseline resolution of BMAA from its potentially confounding structural isomers N-2-aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB) is achieved using the chip-based CE-MS system in less than 1 min. Detection and linearity of response are demonstrated across > 3.5 orders of dynamic range using parallel reaction monitoring (PRM). The lower limit of detection and quantification were calculated for BMAA detection at 40 nM (4.8 ng/mL) and 400 nM (48 ng/mL), respectively. Finally, the strategy was applied to detect BMAA in seafood samples purchased at a local market in Raleigh, NC where their harvest location was known. BMAA was detected in a sea scallop sample. Because the BMAA/stable isotope-labeled 13 C 3 , 15 N 2 -BMAA (SIL-BMAA) ratio in the scallop sample was below the limit of quantification, a semiquantitative analysis of BMAA content was carried out, and BMAA content was estimated to be approximately 820 ng BMAA/1 g of wet scallop tissue. Identification was verified by high mass measurement accuracy of precursor (< 5 ppm) and product ions (< 10 ppm), comigration with SIL-BMAA spike-in standard, and conservation of ion abundance ratios for product ions between BMAA and SIL-BMAA. Interestingly, BMAA was not identified in the free protein fraction but only detected after protein hydrolysis which suggests that BMAA is tightly bound by and/or incorporated into proteins. Graphical abstract Utilization of novel 13C3,15N2-BMAA and chip-based CE-MS/MS for detection and quantification of BMAA in environmental samples.

Laser systems are widely used in mass spectrometry as sample probes and ionization sources. Mid-infrared lasers are particularly suitable for analysis of high water content samples such as animal and plant tissues, using water as a resonantly excited sacrificial matrix. Commercially available mid-IR lasers have historically been bulky and expensive due to cooling requirements. This work presents a novel air-cooled miniature mid-IR laser with adjustable burst-mode output and details an evaluation of its performance for mass spectrometry imaging. The miniature laser was found capable of generating sufficient energy for complete ablation of animal tissue in the context of an IR-MALDESI experiment with exogenously added ice matrix, yielding several hundred confident metabolite identifications. Graphical abstract The use of a novel miniature 2.94 μm burst-mode laser in IR-MALDESI allows for rapid and sensitive mass spectrometry imaging of a whole mouse.

A quantitative mass spectrometry imaging (QMSI) method for absolute quantification of glutathione (GSH) in healthy and cancerous hen ovarian tissues using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is presented. Using this technique, the ion abundance of GSH was normalized to that of a structural analogue, which was sprayed on the slide prior to mounting the tissue sections. This normalization strategy significantly improved the voxel-to-voxel variability; the variability is attributed to the overall ionization process. Subsequently, a series of calibration spots of stable isotope-labeled (SIL) GSH were pipetted on top of the tissue to construct a spatial calibration curve, and calculate the concentration of GSH in both tissue sections. The QMSI results were verified by LC-MS/MS quantification of GSH for the same tissues. GSH was extracted from tissue sections in a slightly acidic buffer and was then alkylated using N-ethylmaleimide to minimize autoxidation of GSH to glutathione disulfide. The alkylated GSH was separated from other contaminants using reversed phase liquid chromatography (RPLC) coupled to a triple quadrupole mass spectrometer, and the z-ion transition of NEM-GSH was used to quantify GSH in each tissue section. While the absolute values obtained using IR-MALDESI QMSI and LC-MS/MS were different, a ∼2-fold increase in the concentration of GSH in cancer tissue compared to the healthy tissue was observed using both techniques. Possible reasons for the difference between absolute concentration values obtained using IR-MALDESI QMSI and LC-MS/MS are also discussed.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitrap's SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13 C, 15 N, and 34 S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms.

The INLIGHT™ strategy for N-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT™ strategy and adopted it for use with O-linked glycans. A central composite design was utilized to find optimized tagging conditions (45% acetic acid, 0.1 μg/μL tag concentration, 37 C, 1.75 h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging of O-linked glycans for the first time. Graphical abstract The INLIGHT™ hydrazide tag was optimized using a central composite design for derivatization of O-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT™ derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.