Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.

Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Effect of lipid extraction and room temperature transportation of bovine oocytes determined by MRM profiling.

Lipids play many important physiological roles in mammalian reproduction, being essential for the acquisition of oocyte competence and post-fertilization embryonic development. Lipid profiling in samples of minute size, such as oocytes, is challenging but has been achieved by mass spectrometry technologies such as multiple reaction monitoring (MRM) profiling. With the goals of further simplifying sample workflow and investigating the influence of pre-analytical conditions, we have evaluated how different extraction methods and transportation of lipid extracts in vacuum and at room temperature impacted the lipid profile of bovine oocytes. Using a comprehensive method, 316 MRMs associated with lipids of 10 different classes were screened in oocyte lipid extracts prepared by 2 extraction methods (one-step methanol addition or Bligh and Dyer) and transporting them in dry ice or at room temperature inside vacuum packages. No changes in the multivariate analysis (PCA) were noticeable due to transportation temperature, while lipid profiles were more affected by the lipid extraction protocol. Sample extraction using pure methanol favored the detection of phospholipids uniformly, while Bligh and Dyer favored the detection of neutral intracellular lipids. Triacylglycerol lipids and free fatty acids yielded decreased abundances when samples were transported at room temperature. We conclude that if samples are submitted to the same lipid extraction protocol and same transportation batch at room temperature coupled with vacuum conditions it is possible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Suspect screening of exogenous compounds using multiple reaction screening (MRM) profiling in human urine samples.

Thousands of chemical compounds produced by industry are dispersed in the human environment widely enough to reach the world population, and the introduction of new chemicals constantly occurs. As new synthetic molecules emerge, rapid analytical workflows for screening possible presence of exogenous compounds in biofluids can be useful as a first pass analysis to detect chemical exposure and guide the development and application of more elaborate LC-MS/MS methods for quantification. In this study, a suspect screening workflow using the multiple reaction monitoring (MRM) profiling method is proposed as a first pass exploratory technique to survey selected exogenous molecules in human urine samples. The workflow was applied to investigate 12 human urine samples using 310 MRMs related to the chemical functionalities of 87 exogenous compounds present in the METLIN database and reported in the literature. A total of 11 MRMs associated with five different compounds were detected in the samples. Product ion scans for the precursor ions of the selected MRMs were acquired as a further identification step for these chemicals. The suspect screening results suggested the presence of five exogenous compounds in the human urine samples analyzed, namely metformin, metoprolol, acetaminophen, paraxanthine and acrylamide. LC-MS/MS was applied as a last step to confirm these results, and the presence of four out of the five targets selected by MRM profiling were corroborated, indicating that this workflow can support the selection of suspect compounds to screen complex samples and guide more time-consuming and specific quantification analyses.

Discovering the N-Terminal Methylome by Repurposing of Proteomic Datasets.

Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation and methylation, on amino acids at the first position. We tested three potentially methylated proteins and confirmed the α-N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.

Shedding Light on the Dynamic Role of the "Target of Rapamycin" Kinase in the Fast-Growing C4 Species Setaria viridis, a Suitable Model for Biomass Crops.

The Target of Rapamycin (TOR) kinase pathway integrates energy and nutrient availability into metabolism promoting growth in eukaryotes. The overall higher efficiency on nutrient use translated into faster growth rates in C4 grass plants led to the investigation of differential transcriptional and metabolic responses to short-term chemical TOR complex (TORC) suppression in the model Setaria viridis. In addition to previously described responses to TORC inhibition (i.e., general growth arrest, translational repression, and primary metabolism reprogramming) in Arabidopsis thaliana (C3), the magnitude of changes was smaller in S. viridis, particularly regarding nutrient use efficiency and C allocation and partitioning that promote biosynthetic growth. Besides photosynthetic differences, S. viridis and A. thaliana present several specificities that classify them into distinct lineages, which also contribute to the observed alterations mediated by TOR. Indeed, cell wall metabolism seems to be distinctly regulated according to each cell wall type, as synthesis of non-pectic polysaccharides were affected in S. viridis, whilst assembly and structure in A. thaliana. Our results indicate that the metabolic network needed to achieve faster growth seems to be less stringently controlled by TORC in S. viridis.

Utilizing Pork Exudate Metabolomics to Reveal the Impact of Aging on Meat Quality.

This study was performed to assess the changes in meat quality and metabolome profiles of meat exudate during postmortem aging. At 24 h postmortem, longissimus lumborum muscles were collected from 10 pork carcasses, cut into three sections, and randomly assigned to three aging period groups (2, 9, and 16 d). Meat quality and chemical analyses, along with the metabolomics of meat exudates using ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) platform, were conducted. Results indicated a declined (p < 0.05) display color stability, and increased (p < 0.05) purge loss, meat tenderness, and lipid oxidation as aging extended. The principal component analysis and hierarchical clustering analysis exhibited distinct clusters of the exudate metabolome of each aging treatment. A total of 39 significantly changed features were tentatively identified via matching them to METLIN database according to their MS/MS information. Some of those features are associated with adenosine triphosphate metabolism (creatine and hypoxanthine), antioxidation (oxidized glutathione and carnosine), and proteolysis (dipeptides and tripeptides). The findings provide valuable information that reflects the meat quality's attributes and could be used as a source of potential biomarkers for predicting aging times and meat quality changes.

A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

Elucidating mechanisms involved in flavor generation of dry-aged beef loins using metabolomics approach.

The present study was conducted to identify flavor-related chemical compounds and to elucidate beef flavor development in response to dry-aging. Paired grass-fed beef loins (n = 18) were obtained at 7 d postmortem, cut into two sections and assigned to 3 aging methods: conventional dry-aging (DA), vacuum packaged wet-aging (WA) and dry-aging in a bag (DW) for 28 days. Following aging, samples were analyzed for UPLC-MS metabolomics, volatile, fatty acid profiling, and consumer sensory comment analysis. Greater number of proteins and nucleotides derived metabolites were liberated in dry-aged samples compared to WA (P < 0.05). In particular, the liberation of gammaglutmayl peptides and glutamine metabolites through the glutathione metabolism were identified. While fatty acid profile was not affected by treatments (P > 0.05), higher concentrations of volatile compounds were found in the dry-aged (P < 0.05). Dry-aging process decreased the presence of terpenoid and steroid lipid group, which could possibly result in reducing undesirable flavor of grass-fed beef.

Harnessing nerve-muscle cell interactions for biomaterials-based skeletal muscle regeneration.

Nerve cells secrete neurotrophic factors that play a critical role in neuronal survival, proliferation, and regeneration. However, their role in regulating myoblast behavior and skeletal muscle repair remains largely unexplored. In the present study, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior under physiologically relevant conditions. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose-dependent manner. We further developed a biomimetic, tunable hydrogel consisting of hyaluronic acid, chondroitin sulfate, and polyethylene glycol as a 3D matrix encapsulating PC12 cells. The hydrogel-encapsulated PC12 cells promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2,088 proteins from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve-secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These experiments provide insights into the nerve-muscle interactions and pave the way for developing advanced biomaterials strategies incorporating nerve cell secretome for accelerated skeletal muscle regeneration.

Multiple reaction monitoring profiling as an analytical strategy to investigate lipids in extracellular vesicles.

Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 µl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 µg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.

Aldol Reactions of Biorenewable Triacetic Acid Lactone Precursor Evaluated Using Desorption Electrospray Ionization Mass Spectrometry High-Throughput Experimentation and Validated by Continuous Flow Synthesis.

Desorption electrospray ionization-mass spectrometry (DESI-MS) was used as a high-throughput experimentation (HTE) tool to rapidly identify derivatives of the biobased platform molecule triacetic acid lactone (TAL). TAL is a platform molecule capable of conversion to a wide range of useful commodity chemicals, agrochemicals, and advanced pharmaceutical intermediates. In the present study, a diverse family of aldol reaction mixtures were prepared in high-density microtiter plates with a liquid handling robot, then printed with a pin tool onto a PTFE surface for analysis by DESI-MS. Our DESI-MS results indicate that aldol products of TAL were obtained for each substrate tested, in good agreement with previously reported TAL reactivity. These HTE experiments also revealed solvent-dependent reactivity trends that facilitated reaction scale up. Our findings suggest that DESI-MS analysis can rapidly inform the selection of optimal reaction conditions from a wide variety of conditions for scale up using continuous synthesis conditions.

Shotgun proteome analysis of seminal plasma differentiate boars by reproductive performance.

There is a need to identify subfertile boars before they enter into the breeding herd. Seminal plasma proteins are essential for normal sperm function and transport and play an important role in fertilization. The objective of this study was to use liquid chromatography tandem mass spectrometry for shotgun proteome analysis to investigate whether differences in boar fertility phenotype can be differentiated by seminal plasma protein abundance. Following 50 breedings, boars were categorized into one of four phenotypes: high farrowing rate and total born (HFHB; n = 9), high farrowing rate with low total born (HFLB; n = 10), low farrowing rate and total born (LFLB; n = 9), and low farrowing rate with high total born (LFHB; n = 4) that were distinct (p < 0.05) from each other by these variables. There were 506 proteins measured in at least one sample across all animals. There were 245 high confidence proteins and 56 were differentially abundant between the high fertility phenotype (HFHB) and at least one of the three subfertile groups. Findings support that seminal plasma protein profiles are distinct between boars with different fertility phenotypes.

High-throughput screening of organic reactions in microdroplets using desorption electrospray ionization mass spectrometry (DESI-MS): hardware and software implementation.

This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer. Analysis rates are up to 1 reaction mixture per second and data are recorded in real time using an ion trap mass spectrometer. Beacon compounds are used to triangulate position on the plate and this allows tandem mass spectrometry (MS/MS) to be performed on confirm products of interest. Custom software allows the user to control the system. It is also used to receive data from the DESI mass spectrometer to screen the spectra for compounds of interest, to perform MS/MS and to save data. This custom software also communicates with the software controlling the fluid handling robot (Biomek i7) as well as the Beckman software used to prepare reaction mixtures and also the software that controls the solvent used as the DESI spray. Data were recorded for N-alkylation, N-acylation and N-sulfonylation reactions in three 8 hour experiments on successive days to establish the ruggedness and repeatability of the system. Repeatability is high (94-97%) over this period with false negative 6% (depending on noise threshold chosen). Plates containing 384 reaction mixtures are analyzed in 7 min by moving the DESI sprayer in steps under the sprayer instead of continuously.

Proteomic identification of aerobic glycolysis as a potential metabolic target for methylglyoxal in adipocytes.

Obesity is often accompanied by metabolic changes in adipocytes that are closely associated with metabolic disease. Although high sugar consumption contributes to obesity, it may also directly affect adipocytes by increasing the rate of glycolysis and formation of the glycolytic by-product methylglyoxal (MG). MG is a reactive dicarbonyl that irreversibly damages proteins and other cellular components. Although the accumulation of MG is clinically associated with hyperglycemia and diabetic complications, a better understanding of how proteins are regulated by MG is needed to evaluate its role in the pathogenesis of metabolic disease. Because adipocytes rely heavily on glycolysis for glucose disposal, we hypothesized that prolonged MG treatment at nontoxic concentrations would impact the landscape of proteins involved in glucose metabolism. To test this hypothesis, we treated 3T3-L1 adipocytes with MG (100 µmol/L) and used comparative proteomics to assess the effects. We identified 25 differentially expressed proteins in adipocytes treated with MG compared to the control. Our results suggested that MG induced metabolic changes typically associated with aerobic glycolysis, including a lowered expression of proteins involved in oxidative metabolism and increased expression of the glycolytic enzymes L-lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The detection of increased lactate secreted into the culture media of adipocytes treated with MG further supported these findings, as did gene expression analysis. In summary, these results indicate MG as a metabolic contributor to aerobic glycolysis in adipocytes, a potential adaptive response to increased glucose flux which over time could lead to permanent metabolic changes.

High-fat-diet induced obesity increases the proportion of linoleic acyl residues in dam serum and milk and in suckling neonate circulation.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes.

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

High-Throughput Experimentation and Continuous Flow Evaluation of Nucleophilic Aromatic Substitution Reactions.

Nucleophilic aromatic substitution (SNAr) reactions were optimized using high-throughput experimentation techniques for execution under flow conditions. A total of 3072 unique reactions were evaluated with an analysis time of ∼3.5 s per reaction using a system that combines a liquid handling robot for reaction mixture preparation with desorption electrospray ionization (DESI) mass spectrometry (MS) for analysis. The reactions were performed in bulk microtiter arrays with and without incubation. In-house developed software was used to process the data and generate heat maps of the results. This information was then used to select the most promising conditions for continuous synthesis under microfluidic reactor conditions. Our results show that this HTE approach provides robust guidance for narrowing the range of conditions needed for optimization of SNAr reactions.

Global Proteomic Analyses of STING-Positive and -Negative Macrophages Reveal STING and Non-STING Differentially Regulated Cellular and Molecular Pathways.

PURPOSE: Cyclic guanosine monophosphate-adenosine monophosphate and other bacterial-derived cyclic di-guanosine monophosphate or cyclic di-adenosine monophosphate trigger innate immune responses through binding to stimulator of interferon genes (STING). Thus in chronic infection, such as in periodontitis, immune cells can be exposed to bacterial DNA and/or cyclic dinucleotides, potentially activating STING to cause inflammation. Thus far the cyclic GMP-AMP synthase-STING- TANK-binding kinase 1 pathway has been well characterized but a global perspective of how the presence or lack of STING affect the proteome is lacking. The aim of this study is to identify macrophage proteins that are affected by STING. EXPERIMENTAL DESIGN: Proteins are extracted from a macrophage cell line harboring STING (RAW-Blue ISG) as well as a STING knockout (STING KO) cell line (RAW-Lucia ISG-KO-STING) and global proteomics analyses are performed. RESULTS: Proteins related to kinase and phosphatase signaling, spliceosome, terpenoid backbone biosynthesis, glycosylation, ubiquitination, and phagocytosis are affected by STING knock out. CONCLUSIONS AND CLINICAL RELEVANCE: STING pathway in macrophages is related to the regulation of several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Moreover, the relation between STING and phagocytosis is demonstrated for the first time. Further validation studies will help identify molecules and pathways that may function as diagnostic or therapeutic targets.