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Quantitative mass spectrometry imaging (qMSI) provides the relative or absolute analyte quantities in a biological specimen in a spatially resolved manner. However, the chemical complexity and physical structure of biological specimens often require one to precisely account for matrix effects in qMSI platforms. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) completely ablates a volume of cryosectioned tissue. This enables the use of a normalization standard that is sprayed underneath the tissue for qMSI applications. Complete sampling has shown to be a significant advantage for qMSI by IR-MALDESI; however, the impact of high tissue heterogeneity has not been systematically studied or quantified. The bias introduced by tissue heterogeneity was investigated by uniformly spraying standards beneath and on top of a whole-body zebrafish section. The quantitative relationship between the signals of the two standards was investigated across this multi-organ model to serve future qMSI experiments by IR-MALDESI and other laser ablation-based sampling methods. The overall ratio between the standards sprayed on top of and beneath the tissue sections remained constant across the entire whole-body section despite significant tissue heterogeneity (e.g., gills, heart, and liver). Additionally, we noted that thinner and/or sucrose-embedded tissues improved these ratios, which will inform future qMSI investigations., Competing Interests: Declarations Animal husbandry Mouse husbandry procedures were approved by the NCSU Institutional Animal Care and Use Committee (IACUC, #19–811-B). Zebrafish husbandry was also in accordance with institutional standard procedures (IACUC, #22–738-B). Competing interests D.C.M. is the interim chief scientific officer and part-owner of MSI Software Solutions, LLC. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Mass spectrometry imaging (MSI) platforms such as infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) are advantageous for a variety of applications, including elucidating the localization of neurotransmitters (NTs) and related molecules with respect to ion abundance across a sample without the need for derivatization or organic matrix application. While IR-MALDESI-MSI conventionally uses a thin exogenous ice matrix to improve signal abundance, it has been previously determined that sucrose embedding without the ice matrix improves detection of lipid species in striatal, coronal mouse brain sections. This work considers components of this workflow to determine the optimal sample preparation and matrix to enhance the detection of NTs and their related metabolites in coronal sections from the striatal region of the mouse brain. The discoveries herein will enable more comprehensive follow-on studies for the investigation of NTs to enrich biological pathways and interpretation related to neurodegenerative diseases and ischemic stroke., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI) conventionally utilizes fresh-frozen biological tissues with an ice matrix to improve the detection of analytes. Sucrose-embedding with paraformaldehyde fixation has demonstrated feasibility as an alternative matrix for analysis by IR-MALDESI by preserving tissue features and enhancing ionization of lipids. However, investigating multi-organ systems provides broader context for a biological study and can elucidate more information about a disease state as opposed to a single organ. Danio rerio, or zebrafish, are model organisms for various disease states and can be imaged as a multi-organ sample to analyze morphological and metabolomic preservation as a result of sample preparation. Herein, whole-body zebrafish were imaged to compare sucrose-embedding with paraformaldehyde fixation against conventional fresh-frozen sample preparation. Serial sections were analyzed with and without an ice matrix to evaluate if sucrose functions as an alternative energy-absorbing matrix for IR-MALDESI applications across whole-body tissues. The resulting four conditions were compared in terms of total putative lipid annotations and category diversity, coverage across the entire m/z range, and ion abundance. Ultimately, sucrose-embedded zebrafish had an increase in putative lipid annotations for the combination of putative annotations with and without the application of an ice matrix relative to fresh-frozen tissues which require the application of an ice matrix. Upon the use of an ice matrix, a greater number of high mass putative lipid annotations (e.g., glycerophospholipids, glycerolipids, and sphingolipids) were identified. Conversely, without an ice matrix, sucrose-embedded sections elucidated more putative annotations in lower molecular weight lipids, including fatty acyls and sterol lipids. Similar to the mouse brain model, sucrose-embedding increased putative lipid annotation and abundance for whole-body zebrafish., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Mass spectrometry imaging (MSI) is an analytical technique that can detect and visualize thousands of m/z values resolved in two- and three-dimensional space. These m/z values lead to hundreds of molecular annotations, including on-tissue and background ions. Discrimination of sample-related analytes from ambient ions conventionally involves manual investigation of each ion heatmap, which requires significant researcher time and effort (for a single tissue image, it can take an hour to determine on-tissue and off-tissue species). Moreover, manual investigation lends itself to subjectivity. Herein, we present the utility of an ion classification tool (ICT) developed using object-based image analysis in MATLAB. The ICT functions by segmenting ion heatmap images into on-tissue and off-tissue objects through binary conversion. The binary images are analyzed and within seconds used to classify the ions as on-tissue or background using a binning approach based on the number of detected objects. In a representative dataset with 50 randomly selected annotations, the ICT was able to accurately classify 45/50 ions as on-tissue or background., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal neurodegenerative disease characterized by progressive loss of motor function with an average survival time of 2-5 years after diagnosis. Due to the lack of signature biomarkers and heterogenous disease phenotypes, a definitive diagnosis of ALS can be challenging. Comprehensive investigation of this disease is imperative to discovering unique features to expedite the diagnostic process and improve diagnostic accuracy. Here, we present untargeted metabolomics by mass spectrometry imaging (MSI) for comparing sporadic ALS (sALS) and C9orf72 positive (C9Pos) post-mortem frontal cortex human brain tissues against a control cohort. The spatial distribution and relative abundance of metabolites were measured by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI for association to biological pathways. Proteomic studies on the same patients were completed via LC-MS/MS in a previous study, and results were integrated with imaging metabolomics results to enhance the breadth of molecular coverage. Utilizing METASPACE annotation platform and MSiPeakfinder, nearly 300 metabolites were identified across the sixteen samples, where 25 were identified as dysregulated between disease cohorts. The dysregulated metabolites were further examined for their relevance to alanine, aspartate, and glutamate metabolism, glutathione metabolism, and arginine and proline metabolism. The dysregulated pathways discussed are consistent with reports from other ALS studies. To our knowledge, this work is the first of its kind, reporting on the investigation of ALS post-mortem human brain tissue analyzed by multiomic MSI.

Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging., (© 2022. The Author(s), under exclusive licence to American Aging Association.)

Dietary fructose, especially in the context of a high-fat western diet, has been linked to type 2 diabetes. Although the effect of fructose on liver metabolism has been extensively studied, a significant portion of the fructose is first metabolized in the small intestine. Here, we report that dietary fat enhances intestinal fructose metabolism, which releases glycerate into the blood. Chronic high systemic glycerate levels induce glucose intolerance by slowly damaging pancreatic islet cells and reducing islet sizes. Our findings provide a link between dietary fructose and diabetes that is modulated by dietary fat., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Native Americans domesticated maize ( Zea mays ssp. mays ) from lowland teosinte parviglumis ( Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 ( HPC1 ), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

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.)

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.

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.)

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.

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.)

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.

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.)

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.

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.

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.

In the field of mass spectrometry imaging, typical experiments involve ionization directly from complex samples with no pre-ionization separation, relying on high resolving power mass analyzers to separate ions of interest. When an ion trapping step is involved in the analysis, the dynamic range of the analysis may be limited by the capacity of the ion trap, which is easily exceeded. To minimize collection of undesired ambient species while maximizing collection of analyte signal, accurate timing between ion generation and collection is a requirement. Here, a method for achieving synchronicity between infrared laser ablation and ion collection on a Q Exactive Plus mass spectrometer is described and demonstrated through measurement of ion accumulation at fixed time points following a laser ablation event with electrospray post-ionization of ablated material. In a model imaging experiment using infrared matrix-assisted laser desorption electrospray ionization, fixing the injection time at the minimum duration required to capture all ions generated by the last laser pulse in a sequence is shown to maximize target ion abundances. Using optimized timing is shown to yield a doubling or better of useful signal compared to previously used parameters. Graphical abstract Illustration of the effects of signal optimization on data quality for a single lipid species (cholesterol) measured from mouse liver tissue.

Background: Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed., Methods: M. oryzae strain 70-15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools., Results: A total of 5498  M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae ., Conclusion: The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

Our greater understanding of the importance of N-linked glycosylation in biological systems has spawned the field of glycomics and development of analytical tools to address the many challenges regarding our ability to characterize and quantify this complex and important modification as it relates to biological function. One of the unmet needs of the field remains a systematic method for characterization of glycans in new biological systems. This study presents a novel workflow for identification of glycans using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy developed in our lab. This consists of monoisotopic mass extraction followed by peak pair identification of tagged glycans from a theoretical library using an in-house program. Identification and relative quantification could then be performed using the freely available bioinformatics tool Skyline. These studies were performed in the biological context of studying the N-linked glycome of differentiating xylem of the poplar tree, a widely studied model woody plant, particularly with respect to understanding lignin biosynthesis during wood formation. Through our workflow, we were able to identify 502 glycosylated proteins including 12 monolignol enzymes and 1 peroxidase (PO) through deamidation glycosite analysis. Finally, our novel semi-automated workflow allowed for rapid identification of 27 glycans by intact mass and by NAT/SIL peak pairing from a library containing 1573 potential glycans, eliminating the need for extensive manual analysis. Implementing Skyline for relative glycan quantification allowed for improved accuracy and precision of quantitative measurements over current processing tools which we attribute to superior algorithms correction for baseline variation and MS1 peak filtering. Graphical abstract Workflow for FANGS-INLIGHT glycosite profiling of plant xylem and monolignol proteins followed by INLIGHT tagging with semi-automated identification of glycans by light-heavy peak pairs. Finally, manual validation and relative quantification was performed in Skyline.

Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of "omics" and "big data." Here, we provide an integrated approach for systematic analysis of the "lipidome" in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos.

The three white perch (Morone americana) vitellogenins (VtgAa, VtgAb, VtgC) were quantified accurately and precisely in the liver, plasma, and ovary during pre-, early-, mid-, and post-vitellogenic oocyte growth using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Western blotting generally mirrored the PC-IDMS results. By PC-IDMS, VtgC was quantifiable in pre-vitellogenic ovary tissues and VtgAb was quantifiable in pre-vitellogenic liver tissues however, neither protein was detected by western blotting in these respective tissues at this time point. Immunohistochemistry indicated that VtgC was present within pre-vitellogenic oocytes and localized to lipid droplets within vitellogenic oocytes. Affinity purification coupled to tandem mass spectrometry using highly purified VtgC as a bait protein revealed a single specific interacting protein (Y-box binding protein 2a-like [Ybx2a-like]) that eluted with suramin buffer and confirmed that VtgC does not bind the ovary vitellogenin receptors (LR8 and Lrp13). Western blotting for LR8 and Lrp13 showed that both receptors were expressed during vitellogenesis with LR8 and Lrp13 expression highest in early- and mid-vitellogenesis, respectively. The VtgAa within the ovary peaked during post-vitellogenesis, while VtgAb peaked during early-vitellogenesis in both white perch and the closely related striped bass (M. saxatilis). The VtgC was steadily accumulated by oocytes beginning during pre-vitellogenesis and continued until post-vitellogenesis and its composition varies widely between striped bass and white perch. In striped bass, the VtgC accounted for 26% of the vitellogenin-derived egg yolk, however in the white perch it comprised only 4%. Striped bass larvae have an extended developmental window and these larvae have yolk stores that may enable them to survive in the absence of food for twice as long as white perch after hatch. Thus, the VtgC may play an integral role in providing nutrients to late stage fish larvae prior to the onset of exogenous feeding and its composition in the egg yolk may relate to different early life histories among this diverse group of animals.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn(2+)-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼ 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser(123) or Ser(125) in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser(123) or Ser(125) and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser(123) phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser(123) or Ser(125). PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

Persistent HIV replication within active viral reservoirs may be caused by inadequate antiretroviral penetration. Here, we used mass spectrometry imaging with infrared matrix-assisted laser desorption-electrospray ionization to quantify the distribution of efavirenz within tissues from a macaque dosed orally to a steady state. Intratissue efavirenz distribution was heterogeneous, with the drug concentrating in the lamina propria of the colon, the primary follicles of lymph nodes, and the brain gray matter. These are the first imaging data of an antiretroviral drug in active viral reservoirs., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Human embryonic stem cells (hESCs) have been routinely treated with bone morphogenetic protein and/or inhibitors of activin/nodal signaling to obtain cells that express trophoblast markers. Trophoblasts can terminally differentiate to either extravillous trophoblasts or syncytiotrophoblasts. The signaling pathways that govern the terminal fate of these trophoblasts are not understood. We show that activin/nodal signaling switches the terminal fate of these hESC-derived trophoblasts. Inhibition of activin/nodal signaling leads to formation of extravillous trophoblast, whereas loss of activin/nodal inhibition leads to the formation of syncytiotrophoblasts. Also, the ability of hESCs to form bona fide trophoblasts has been intensely debated. We have examined hESC-derived trophoblasts in the light of stringent criteria that were proposed recently, such as hypomethylation of the ELF5-2b promoter region and down-regulation of HLA class I antigens. We report that trophoblasts that possess these properties can indeed be obtained from hESCs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2 ± 1.8 μg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4 ± 2.8 μg/gtissue.

Mass spectrometry imaging (MSI) allows for the direct and simultaneous analysis of the spatial distribution of molecular species from sample surfaces such as tissue sections. One of the goals of MSI is monitoring the distribution of compounds at the cellular resolution in order to gain insights about the biology that occurs at this spatial level. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging of cervical tissue sections was performed using a spot-to-spot distance of 10 μm by utilizing the method of oversampling, where the target plate is moved by a distance that is less than the desorption radius of the laser. In addition to high spatial resolution, high mass accuracy (±1 ppm) and high mass resolving power (140,000 at m/z = 200) were achieved by coupling the IR-MALDESI imaging source to a hybrid quadrupole Orbitrap mass spectrometer. Ion maps of cholesterol in tissues were generated from voxels containing <1 cell, on average. Additionally, the challenges of imaging at the cellular level in terms of loss of sensitivity and longer analysis time are discussed.

Determining the distribution of a drug and its metabolites within tissue is a key facet of evaluating drug candidates. Drug distribution can have a significant implication in appraising drug efficacy and potential toxicity. The specificity and sensitivity of mass spectrometry imaging (MSI) make it a perfect complement to the analysis of drug distributions in tissue. The detection of lapatinib as well as several of its metabolites in liver tissue was determined by MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to high resolving power Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. IR-MALDESI required minimal sample preparation while maintaining high sensitivity. The effect of the electrospray solvent composition on IR-MALDESI MSI signal from tissue analysis was investigated and an empirical comparison of IR-MALDESI and UV-MALDI for MSI analysis is also presented.

Trace evidence is a significant portion of forensic cases. Textile fibers are a common form of trace evidence that are gaining importance in criminal cases. Currently, qualitative techniques that do not yield structural information are primarily used for fiber analysis, but mass spectrometry is gaining an increasing role in this field. Mass spectrometry yields more quantitative structural information about the dye and polymer that can be used for more conclusive comparisons. Matrix-assisted laser desorption electrospray ionization (MALDESI) is a hybrid ambient ionization source being investigated for use in mass spectrometric fiber analysis. In this manuscript, IR-MALDESI was used as a source for mass spectrometry imaging (MSI) of a dyed nylon fiber cluster and single fiber. Information about the fiber polymer as well as the dye were obtained from a single fiber which was on the order of 10 μm in diameter. These experiments were performed directly from the surface of a tape lift of the fiber with a background of extraneous fibers.

Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

In a number of plant species, downregulation of 4-coumaric acid: coenzyme A ligase (4CL) can reduce lignin content. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes Ptr4CL3 and Ptr4CL5 catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA: shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only formation of 4-coumaroyl-CoA. 4-coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Estimates of metabolic flux in wildtype and PtrC3H3 downregulated P. trichocarpa transgenics have been made using LC-MS/MS based absolute protein and metabolite quantification, mass action kinetics and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate., (© Crown copyright 2014.)

As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.