Publications by authors named "Joshua J Coon"

273 Publications

Multi-Omic Single-Shot Technology for Integrated Proteome and Lipidome Analysis.

Anal Chem 2021 Feb 22. Epub 2021 Feb 22.

Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Mass spectrometry (MS) serves as the centerpiece technology for proteome, lipidome, and metabolome analysis. To gain a better understanding of the multifaceted networks of myriad regulatory layers in complex organisms, integration of different multiomic layers is increasingly performed, including joint extraction methods of diverse biomolecular classes and comprehensive data analyses of different omics. Despite the versatility of MS systems, fractured methodology drives nearly all MS laboratories to specialize in analysis of a single ome at the exclusion of the others. Although liquid chromatography-mass spectrometry (LC-MS) analysis is similar for different biomolecular classes, the integration on the instrument level is lagging behind. The recent advancements in high flow proteomics enable us to take a first step towards integration of protein and lipid analysis. Here, we describe a technology to achieve broad and deep coverage of multiple molecular classes simultaneously through multi-omic single-shot technology (MOST), requiring only one column, one LC-MS instrument, and a simplified workflow. MOST achieved great robustness and reproducibility. Its application to a study consisting of 20 conditions revealed 2842 protein groups and 325 lipids and potential molecular relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.0c04764DOI Listing
February 2021

Unique inflammatory profile is associated with higher SARS-CoV-2 acute respiratory distress syndrome (ARDS) mortality.

Am J Physiol Regul Integr Comp Physiol 2021 Jan 12. Epub 2021 Jan 12.

Medicine, Albany Medical College, United States.

The COVID19 pandemic has caused more than a million of deaths worldwide, primarily due to complications from COVID19-associated acute respiratory distress syndrome (ARDS). Controversy surrounds the circulating cytokine/chemokine profile of COVID19-associated ARDS, with some groups suggesting that it is similar to non-COVID19 ARDS patients and others observing substantial differences. Moreover, while a hyperinflammatory phenotype associates with higher mortality in non-COVID19 ARDS, there is little information on the inflammatory landscape's association with mortality in COVID19 ARDS patients. Even though the circulating leukocytes' transcriptomic signature has been associated with distinct phenotypes and outcomes in critical illness including ARDS, it is unclear whether the mortality-associated inflammatory mediators from COVID19 patients are transcriptionally regulated in the leukocyte compartment. Here, we conducted a prospective cohort study of 41 mechanically ventilated patients with COVID19 infection using highly calibrated methods to define the levels of plasma cytokines/chemokines and their gene expressions in circulating leukocytes. Plasma IL1RA and IL8 were found positively associated with mortality while RANTES and EGF negatively associated with that outcome. However, the leukocyte gene expression of these proteins had no statistically significant correlation with mortality. These data suggest a unique inflammatory signature associated with severe COVID19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpregu.00324.2020DOI Listing
January 2021

Global Phosphoproteome Analysis Using High-Field Asymmetric Waveform Ion Mobility Spectrometry on a Hybrid Orbitrap Mass Spectrometer.

Anal Chem 2020 12 3;92(24):15959-15967. Epub 2020 Dec 3.

National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15-20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.0c03415DOI Listing
December 2020

Quantitative shotgun proteome analysis by direct infusion.

Nat Methods 2020 12 23;17(12):1222-1228. Epub 2020 Nov 23.

Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA.

Liquid chromatography-mass spectrometry (LC-MS) delivers sensitive peptide analysis for proteomics but requires extensive analysis time, reducing throughput. Here, we demonstrate that gas-phase peptide separation instead of LC enables fast proteome analysis. Using direct infusion-shotgun proteome analysis (DI-SPA) by data-independent acquisition mass spectrometry (DIA-MS), we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins per second). We show the utility of this technology in performing a complex multifactorial proteomic study of interactions between nutrients, genotype and mitochondrial toxins in a collection of cultured human cells. More than 45,000 quantitative protein measurements from 132 samples were achieved in only ~4.4 h of MS data collection. Enabling fast, unbiased proteome quantification without LC, DI-SPA offers an approach to boost throughput, critical to drug and biomarker discovery studies that require analysis of thousands of proteomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-020-00999-zDOI Listing
December 2020

Argonaut: A Web Platform for Collaborative Multi-omic Data Visualization and Exploration.

Patterns (N Y) 2020 Oct;1(7)

Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Researchers now generate large multi-omic datasets using increasingly mature mass spectrometry techniques at an astounding pace, facing new challenges of "Big Data" dissemination, visualization, and exploration. Conveniently, web-based data portals accommodate the complexity of multi-omic experiments and the many experts involved. However, developing these tailored companion resources requires programming expertise and knowledge of web server architecture-a substantial burden for most. Here, we describe Argonaut, a simple, code-free, and user-friendly platform for creating customizable, interactive data-hosting websites. Argonaut carries out real-time statistical analyses of the data, which it organizes into easily sharable projects. Collaborating researchers worldwide can explore the results, visualized through popular plots, and modify them to streamline data interpretation. Increasing the pace and ease of access to multi-omic data, Argonaut aims to propel discovery of new biological insights. We showcase the capabilities of this tool using a published multi-omics dataset on the large mitochondrial protease deletion collection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.patter.2020.100122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641515PMC
October 2020

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

Genetics 2020 Dec 27;216(4):1117-1136. Epub 2020 Oct 27.

Department of Medical Genetics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706

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 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, , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/ , and / flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/ flies relative to +/+ and / flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.120.303776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768241PMC
December 2020

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

Cell Syst 2021 01 8;12(1):23-40.e7. Epub 2020 Oct 8.

Division of Pulmonary and Critical Care Medicine, Albany Medical Center, Albany, NY 12208, USA; Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY 12208, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2020.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543711PMC
January 2021

Mapping Physiological ADP-Ribosylation Using Activated Ion Electron Transfer Dissociation.

Cell Rep 2020 09;32(12):108176

Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508052PMC
September 2020

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

Nat Metab 2020 10 21;2(10):1149-1162. Epub 2020 Sep 21.

Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-020-00278-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572687PMC
October 2020

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase.

PLoS Pathog 2020 09 3;16(9):e1008841. Epub 2020 Sep 3.

Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, United States of America.

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494117PMC
September 2020

Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose.

PLoS One 2020 14;15(8):e0226235. Epub 2020 Aug 14.

DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226235PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428164PMC
September 2020

Optical Fiber-Enabled Photoactivation of Peptides and Proteins.

Anal Chem 2020 09 24;92(18):12363-12370. Epub 2020 Aug 24.

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Photoactivation and photodissociation have long proven to be useful tools in tandem mass spectrometry, but implementation often involves cumbersome and potentially dangerous configurations. Here, we redress this problem by using a fiber-optic cable to couple an infrared (IR) laser to a mass spectrometer for robust, efficient, and safe photoactivation experiments. Transmitting 10.6 μm IR photons through a hollow-core fiber, we show that such fiber-assisted activated ion-electron transfer dissociation (AI-ETD) and IR multiphoton dissociation (IRMPD) experiments can be carried out as effectively as traditional mirror-based implementations. We report on the transmission efficiency of the hollow-core fiber for conducting photoactivation experiments and perform various intact protein and peptide analyses to illustrate the benefits of fiber-assisted AI-ETD, namely, a simplified system for irradiating the two-dimensional linear ion trap volume concurrent with ETD reactions to limit uninformative nondissociative events and thereby amplify sequence coverage. We also describe a calibration scheme for the routine analysis of IR laser alignment and power through the fiber and into the dual cell quadrupolar linear ion trap. In all, these advances allow for a more robust, straightforward, and safe instrumentation platform, permitting implementation of AI-ETD and IRMPD on commercial mass spectrometers and broadening the accessibility of these techniques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.0c02087DOI Listing
September 2020

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

medRxiv 2020 Jul 19. Epub 2020 Jul 19.

Division of Pulmonary and Critical Care Medicine, Albany Medical Center, Albany, NY, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.07.17.20156513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388490PMC
July 2020

Constructing and deconstructing GATA2-regulated cell fate programs to establish developmental trajectories.

J Exp Med 2020 Nov;217(11)

University of Wisconsin-Madison Blood Research Program, Department of Cell and Regenerative Biology, Wisconsin Institutes for Medical Research, University of Wisconsin School of Medicine and Public Health, Madison, WI.

Stem and progenitor cell fate transitions constitute key decision points in organismal development that enable access to a developmental path or actively preclude others. Using the hematopoietic system, we analyzed the relative importance of cell fate-promoting mechanisms versus negating fate-suppressing mechanisms to engineer progenitor cells with multilineage differentiation potential. Deletion of the murine Gata2-77 enhancer, with a human equivalent that causes leukemia, downregulates the transcription factor GATA2 and blocks progenitor differentiation into erythrocytes, megakaryocytes, basophils, and granulocytes, but not macrophages. Using multiomics and single-cell analyses, we demonstrated that the enhancer orchestrates a balance between pro- and anti-fate circuitry in single cells. By increasing GATA2 expression, the enhancer instigates a fate-promoting mechanism while abrogating an innate immunity-linked, fate-suppressing mechanism. During embryogenesis, the suppressing mechanism dominated in enhancer mutant progenitors, thus yielding progenitors with a predominant monocytic differentiation potential. Coordinating fate-promoting and -suppressing circuits therefore averts deconstruction of a multifate system into a monopotent system and maintains critical progenitor heterogeneity and functionality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20191526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596813PMC
November 2020

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

mSystems 2020 Jul 21;5(4). Epub 2020 Jul 21.

Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA

is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the genome is a current barrier to both fundamental studies of and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5' and 3' ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5' end. Our results revealed that σ -35 and -10 promoter elements closely resemble canonical -35 and -10 elements, with one notable exception: the -10 element lacks the highly conserved -7 thymine observed in and other previously characterized σ promoters. The σ promoters of another alphaproteobacterium, , similarly lack the conservation of -7 thymine in their -10 elements. Our results anchor the development of as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. Efforts to rationally engineer synthetic pathways in are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to This information is vital as researchers continue to develop for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSystems.00250-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566282PMC
July 2020

Discovering How Heme Controls Genome Function Through Heme-omics.

Cell Rep 2020 06;31(13):107832

Department of Cell and Regenerative Biology, UW-Madison Blood Research Program, Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. Electronic address:

Protein ensembles control genome function by establishing, maintaining, and deconstructing cell-type-specific chromosomal landscapes. A plethora of small molecules orchestrate cellular functions and therefore may link physiological processes with genome biology. The metabolic enzyme and hemoglobin cofactor heme induces proteolysis of a transcriptional repressor, Bach1, and regulates gene expression post-transcriptionally. However, whether heme controls genome function broadly or through prescriptive actions is unclear. Using assay for transposase-accessible chromatin sequencing (ATAC-seq), we establish a heme-dependent chromatin atlas in wild-type and mutant erythroblasts lacking enhancers that confer normal heme synthesis. Amalgamating chromatin landscapes and transcriptomes in cells with sub-physiological heme and post-heme rescue reveals parallel Bach1-dependent and Bach1-independent mechanisms that target heme-sensing chromosomal hotspots. The hotspots harbor a DNA motif demarcating heme-regulated chromatin and genes encoding proteins not known to be heme regulated, including metabolic enzymes. The heme-omics analysis establishes how an essential biochemical cofactor controls genome function and cellular physiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382780PMC
June 2020

Top-Down Characterization of an Intact Monoclonal Antibody Using Activated Ion Electron Transfer Dissociation.

Anal Chem 2020 08 16;92(15):10246-10251. Epub 2020 Jul 16.

Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds-making it attractive for mAb characterization-it can be less effective for precursors having high / values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.0c00705DOI Listing
August 2020

Phosphoproteome Response to Dithiothreitol Reveals Unique Shared Features of Stress Responses.

J Proteome Res 2020 08 13;19(8):3405-3417. Epub 2020 Jul 13.

Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

To cope with sudden changes in the external environment, the budding yeast orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking or MAPK kinases and , before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. These results have implications for the breadth of signaling programs that can give rise to common stress response signatures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c00253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646510PMC
August 2020

Higher plasma levels of Chemokine CCL19 are associated with poor SARS-CoV-2 acute respiratory distress syndrome (ARDS) outcomes.

medRxiv 2020 May 22. Epub 2020 May 22.

Division of Pulmonary and Critical Care Medicine, Albany Medical College, Albany, NY.

COVID19 pandemic has so far caused over three hundred thousand deaths worldwide, primarily due to complications from SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). While an ARDS-driven hyperinflammatory phenotype is associated with higher mortality in non-COVID patients, there is little information on how cytokines and chemokines expressions correlate with clinical outcomes in COVID19 patients. We prospectively enrolled a cohort of 41 patients with acute respiratory distress syndrome on mechanical ventilation. Patients blood was obtained at enrollment and outcome measures were liberation from mechanical ventilation and hospital-free days. We determined the expression levels of 44 circulating cytokines/chemokines and found 13 of them associated with worse outcomes. After correcting for multiple comparisons/false discovery rate, only one chemokine (CCL19) remained significantly associated with outcomes (p=0.009). Although not described in association with COVID19, this chemokine was previously found elevated in an animal model of SARS-CoV. Moreover, CCL19 seems to be relevant for bronchus-associated lymphoid tissue (BALT) maintenance and for lung immunity to influenza virus. While this finding requires corroboration, CCL19 determination could facilitate early identification COVID19-ARDS patients at higher risk of death and be novel target for immunotherapy in this setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.21.20051300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273283PMC
May 2020

Post-Translation Regulation of Influenza Virus Replication.

Annu Rev Virol 2020 09 26;7(1):167-187. Epub 2020 May 26.

Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA; email:

Influenza virus exploits cellular factors to complete each step of viral replication. Yet, multiple host proteins actively block replication. Consequently, infection success depends on the relative speed and efficacy at which both the virus and host use their respective effectors. Post-translational modifications (PTMs) afford both the virus and the host means to readily adapt protein function without the need for new protein production. Here we use influenza virus to address concepts common to all viruses, reviewing how PTMs facilitate and thwart each step of the replication cycle. We also discuss advancements in proteomic methods that better characterize PTMs. Although some effectors and PTMs have clear pro- or antiviral functions, PTMs generally play regulatory roles to tune protein functions, levels, and localization. Synthesis of our current understanding reveals complex regulatory schemes where the effects of PTMs are time and context dependent as the virus and host battle to control infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-virology-010320-070410DOI Listing
September 2020

A bacterial biosynthetic pathway for methylated furan fatty acids.

J Biol Chem 2020 07 20;295(29):9786-9801. Epub 2020 May 20.

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in 2.4.1 and CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (1012)-11-methyloctadeca-10,12-dienoic acid (11Me-10,12-18:2), produced from (11)-methyloctadeca-11-enoic acid (11Me-12-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-1012-18:2 fatty acid phospholipid chain as a substrate. We discovered that also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380195PMC
July 2020

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

ChemSusChem 2020 Apr 14;13(8):1922. Epub 2020 Apr 14.

DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, 53726, USA.

Invited for this month's cover is the research team from the D.O.E. Great Lake Bioenergy Research Center (GLBRC) at the University of Wisconsin-Madison. The cover image shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the liquid fuels and co-products, is assessed, can we identify the key parameters needed to design an economically viable biorefinery-based economy. Cover art by Chelsea Mamott. The Full Paper itself is available at 10.1002/cssc.201903345.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cssc.202000820DOI Listing
April 2020

Tailoring a Global Iron Regulon to a Uropathogen.

mBio 2020 03 24;11(2). Epub 2020 Mar 24.

Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA

Pathogenicity islands and plasmids bear genes for pathogenesis of various pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the mutant. The increased amino acid demand of the mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O to produce aerobactin. Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic (UPEC) to that of nonpathogenic K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other pathotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00351-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157518PMC
March 2020

Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes.

Proc Natl Acad Sci U S A 2020 04 23;117(14):7764-7775. Epub 2020 Mar 23.

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143;

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of -linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1917947117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148585PMC
April 2020

Quantitative capillary zone electrophoresis-mass spectrometry reveals the N-glycome developmental plan during vertebrate embryogenesis.

Mol Omics 2020 06;16(3):210-220

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.

Glycans are known to be involved in many biological processes, while little is known about the expression of N-glycans during vertebrate development. We now report the first quantitative studies of both the expression of N-linked glycans at six early development stages and the expression of N-glycosylated peptides at two early development stages in Xenopus laevis, the African clawed frog. N-Glycans were labeled with isobaric tandem mass tags, pooled, separated by capillary electrophoresis, and characterized using tandem mass spectrometry. We quantified 110 N-glycan compositions that spanned four orders of magnitude in abundance. Capillary electrophoresis was particularly useful in identifying charged glycans; over 40% of the observed glycan compositions were sialylated. The glycan expression was relatively constant until the gastrula-neurula transition (developmental stage 13), followed by massive reprogramming. An increase in oligomannosidic and a decrease in the paucimannosidic and phosphorylated oligomannosidic glycans were observed at the late tailbud stage (developmental stage 41). Two notable and opposing regulation events were detected for sialylated glycans. LacdiNAc and Lewis antigen features distinguished down-regulated sialylation from up-regulated species. The level of Lewis antigen decreased at later stages, which was validated by Aleuria aurantia lectin (AAL) and Ulex europaeus lectin (UEA-I) blots. We also used HPLC coupled with tandem mass spectrometry to identify 611 N-glycosylation sites on 350 N-glycoproteins at the early stage developmental stage 1 (fertilized egg), and 1682 N-glycosylation sites on 1023 N-glycoproteins at stage 41 (late tailbud stage). Over two thirds of the N-glycoproteins identified in the late tailbud stage are associated with neuron projection morphogenesis, suggesting a vital role of the N-glycome in neuronal development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0mo00005aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299754PMC
June 2020

Ribonucleic Acid Sequence Characterization by Negative Electron Transfer Dissociation Mass Spectrometry.

Anal Chem 2020 03 5;92(6):4436-4444. Epub 2020 Mar 5.

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Modified oligonucleotides represent a promising avenue for drug development, with small interfering RNAs (siRNA) and microRNAs gaining traction in the therapeutic market. Mass spectrometry (MS)-based analysis offers many benefits for characterizing modified nucleic acids. Negative electron transfer dissociation (NETD) has proven valuable in sequencing oligonucleotide anions, particularly because it can retain modifications while generating sequence-informative fragments. We show that NETD can be successfully implemented on a widely available quadrupole-Orbitrap-linear ion trap mass spectrometer that uses a front-end glow discharge source to generate radical fluoranthene reagent cations. We characterize both unmodified and modified ribonucleic acids and present the first application of activated-ion negative electron transfer dissociation (AI-NETD) to nucleic acids. AI-NETD achieved 100% sequence coverage for both a 6-mer (5'-rGmUrArCmUrG-3') with 2'--methyl modifications and a 21-mer (5'-rCrArUrCrCrUrCrUrArGrArGrGrArUrArGrArArUrG-3'), the luciferase antisense siRNA. Both NETD and AI-NETD afforded complete sequence coverage of these molecules while maintaining a relatively low degree of undesired base-loss products and internal products relative to collision-based methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.9b05388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161943PMC
March 2020

A Genetic Toggle for Chemical Control of Individual Plk1 Substrates.

Cell Chem Biol 2020 03 3;27(3):350-362.e8. Epub 2020 Feb 3.

Department of Medicine, Division of Hematology/Oncology, University of Wisconsin, 1111 Highland Avenue, WIMR 6059, Madison, WI 53705, USA; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI 53705, USA; Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53705, USA. Electronic address:

Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1 (L197F) and Plk1 (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1 but not Plk1 sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2020.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239509PMC
March 2020

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

ChemSusChem 2020 Apr 11;13(8):2012-2024. Epub 2020 Mar 11.

DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, 53726, USA.

The hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) add diversity to the portfolio of products produced by using grass-fed lignocellulosic biorefineries. The level of lignin-bound pCA in Zea mays was modified by the alteration of p-coumaroyl-CoA monolignol transferase expression. The biomass was processed in a lab-scale alkaline-pretreatment biorefinery process and the data were used for a baseline technoeconomic analysis to determine where to direct future research efforts to couple plant design to biomass utilization processes. It is concluded that future plant engineering efforts should focus on strategies that ramp up accumulation of one type of hydroxycinnamate (pCA or FA) predominantly and suppress that of the other. Technoeconomic analysis indicates that target extraction titers of one hydroxycinnamic acid need to be >50 g kg biomass, at least five times higher than observed titers for the impure pCA/FA product mixture from wild-type maize. The technical challenge for process engineers is to develop a viable process that requires more than 80 % reduction of the isolation costs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cssc.201903345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217007PMC
April 2020

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

Autophagy 2020 12 2;16(12):2219-2237. Epub 2020 Feb 2.

Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati , Cincinnati, OH, USA.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes ( or ) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2020.1719746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751563PMC
December 2020