Improved Mass Accuracy and Precision for Multi-Attribute Methods Using a New Internally Calibrated High Resolution Orbitrap Mass Detector.

In the development of biotherapeutics, a thorough understanding of a molecule's product quality attributes (PQAs) and their effect on structure-function relationships and long-term stability is essential for ensuring the safety and efficacy of the product. First published in 2015, the multi-attribute method (MAM), based on LC-MS peptide mapping and automation principles, can be used to support biotherapeutic process and product development. The MAM provides simultaneous site-specific detection, identification, quantitation, and quality control monitoring of selected PQAs. In this article, a low-maintenance MAM-ready mass detector with a small footprint was evaluated for its ability to monitor PQAs on proteolytically digested proteins with high mass accuracy and precision. Optimized source parameters enable robust relative quantitation of attributes with high sensitivity and minimal in-source fragmentation. A combination of a built-in one-point mass calibration procedure prior to data acquisition and Scan-to-Scan on-the-fly mass correction allows monitoring of most peptides for at least 54 days with sub-1 ppm mass accuracies at high-resolution (180,000 at m/z 200). This enables the use of <3 ppm mass tolerances for peptide monitoring, supporting high method specificity and robustness. LC-MS based MAM data from this instrument compares well to data collected by earlier MAM systems and conventional HPLC profile-based drug substance release assays.

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis.

BACKGROUND: Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism's vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. RESULTS: We utilized a systems-level approach comprising LC-MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis' CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology-including cell swelling and formation of intracellular protein aggregates -when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis' primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. CONCLUSIONS: This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Systematic Analysis of Metabolic Bottlenecks in the Methylerythritol 4-Phosphate (MEP) Pathway of Zymomonas mobilis.

Zymomonas mobilis is an industrially relevant aerotolerant anaerobic bacterium that can convert up to 96% of consumed glucose to ethanol. This highly catabolic metabolism could be leveraged to produce isoprenoid-based bioproducts via the methylerythritol 4-phosphate (MEP) pathway, but we currently have limited knowledge concerning the metabolic constraints of this pathway in Z. mobilis. Here, we performed an initial investigation of the metabolic bottlenecks within the MEP pathway of Z. mobilis using enzyme overexpression strains and quantitative metabolomics. Our analysis revealed that 1-deoxy-d-xylulose 5-phosphate synthase (DXS) represents the first enzymatic bottleneck in the Z. mobilis MEP pathway. DXS overexpression triggered large increases in the intracellular levels of the first five MEP pathway intermediates, of which the buildup in 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) was the most substantial. The combined overexpression of DXS, 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (IspG), and HMBDP reductase (IspH) mitigated the bottleneck at MEcDP and mobilized carbon to downstream MEP pathway intermediates, indicating that IspG and IspH activity become the primary pathway constraints during DXS overexpression. Finally, we overexpressed DXS with other native MEP enzymes and a heterologous isoprene synthase and showed that isoprene can be used as a carbon sink in the Z. mobilis MEP pathway. By revealing key bottlenecks within the MEP pathway of Z. mobilis, this study will aid future engineering efforts aimed at developing this bacterium for industrial isoprenoid production. IMPORTANCE Engineered microorganisms have the potential to convert renewable substrates into biofuels and valuable bioproducts, which offers an environmentally sustainable alternative to fossil-fuel-derived products. Isoprenoids are a diverse class of biologically derived compounds that have commercial applications as various commodity chemicals, including biofuels and biofuel precursor molecules. Thus, isoprenoids represent a desirable target for large-scale microbial generation. However, our ability to engineer microbes for the industrial production of isoprenoid-derived bioproducts is limited by an incomplete understanding of the bottlenecks in the biosynthetic pathway responsible for isoprenoid precursor generation. In this study, we combined genetic engineering with quantitative analyses of metabolism to examine the capabilities and constraints of the isoprenoid biosynthetic pathway in the industrially relevant microbe Zymomonas mobilis. Our integrated and systematic approach identified multiple enzymes whose overexpression in Z. mobilis results in an increased production of isoprenoid precursor molecules and mitigation of metabolic bottlenecks.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

Rapid Targeted Quantitation of Protein Overexpression with Direct Infusion Shotgun Proteome Analysis (DISPA-PRM).

While much effort has been placed on comprehensive quantitative proteome analysis, certain applications demand the measurement of only a few target proteins from complex systems. Traditional approaches to targeted proteomics rely on nanoliquid chromatography (nLC) and targeted mass spectrometry (MS) methods, e.g., parallel reaction monitoring (PRM). However, the time requirement for nLC can limit the throughput of targeted proteomics. To achieve rapid and high-throughput targeted methods, here we show that nLC separations can be eliminated and replaced with direct infusion shotgun proteome analysis (DISPA) using high-field asymmetric waveform ion mobility spectrometry (FAIMS) with PRM. We demonstrate the application of DISPA-PRM for rapid targeted quantification of bacterial enzymes utilized in the production of biofuels by monitoring temporal expression in 72 metabolically engineered bacterial cultures in less than 2.5 h, with a measured dynamic range >1200-fold. We conclude that DISPA-PRM presents a valuable innovative tool with results comparable to nLC-MS/MS, enabling fast and rapid detection of targeted proteins in complex mixtures.

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis.

Zymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production. IMPORTANCE Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol. Developing microbial systems to generate advanced biofuels requires metabolic engineering to reroute carbon away from ethanol and other native products and toward desired pathways, such as the MEP pathway for isoprenoid biosynthesis. However, rational engineering of microbial metabolism relies on understanding metabolic control points, in terms of both enzyme activity and thermodynamic favorability. In Z. mobilis, the factors that control glycolytic rates, ethanol production, and isoprenoid production are still not fully understood. In this study, we performed metabolomic, proteomic, and thermodynamic analysis of Z. mobilis during N2 fixation. This analysis identified key changes in metabolite levels, enzyme abundance, and glycolytic thermodynamic favorability that occurred during changes in NH4+ availability, helping to inform future efforts in metabolic engineering.

Large-Scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity.

Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish.

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4-8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldel on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel , and Reldel /Reldel flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Reldel flies relative to +/+ and Reldel /Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

A large-scale genome-lipid association map guides lipid identification.

Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.

Large-scale Multi-omic Analysis of COVID-19 Severity.

We performed RNA-Seq and high-resolution mass spectrometry on 128 blood samples from COVID-19 positive and negative patients with diverse disease severities. Over 17,000 transcripts, proteins, metabolites, and lipids were quantified and associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a comparative analysis with published data and a machine learning approach for prediction of COVID-19 severity.

Maximizing Tandem Mass Spectrometry Acquisition Rates for Shotgun Proteomics.

Advances in tandem mass spectrometry (MS/MS) acquisition rate have steadily led to increased performance in shotgun proteomics experiments. To that end, contemporary mass spectrometers are outfitted with multiple analyzers allowing for the simultaneous collection of survey (MS1) and MS/MS spectra. In the latest generation Orbitrap hybrid, MS/MS scans can be acquired at a high rate using the dual cell linear ion trap analyzer, all while the next precursor is being dissociated in a collision cell and a MS1 scan is occurring in the Orbitrap. Often overlooked in these experiments is that the ion trap scan duration is highly variable and dependent upon precursor mass. Here, we examine the use of various static mass-to-charge ratio scan ranges for ion trap MS/MS acquisition and determine performance relative to conventional dynamic mass-to-charge ratio range scanning. We demonstrate that a fixed mass-to-charge ratio scan range can generate 12% more MS/MS scans and more unique peptide identifications as compared to the standard dynamic approach, respectively.

Metabolic Remodeling during Biofilm Development of Bacillus subtilis.

Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling.

Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes, and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide molecular insights into mitochondrial protein functions.