Search

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid-chromatography coupled to mass spectrometry (LCMS) has been widely used to characterize post-translational modifications in proteins including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogenous glycans at the intact protein level. Herein, we evaluated several online separation techniques: 1. C2 reverse-phase liquid chromatography (RPLC), 2. capillary zone electrophoresis (CZE), and 3. acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down MS. Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation, N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogenous glycoproteins for facile comparison of biosimilars in quality control applications.

An understanding of the ethical underpinnings of human subjects research that involves some risk to participants without anticipated direct clinical benefit-such as the kidney biopsy procedure as part of the Kidney Precision Medicine Project (KPMP)-requires a critical examination of the risks as well as the diverse set of countervailing potential benefits to participants. This kind of deliberation has been foundational to the development and conduct of the KPMP. Herein, we use illustrative features of this research paradigm to develop a more comprehensive conceptualization of the types of benefits that may be important to research participants, including respecting pluralistic values, supporting the opportunity to act altruistically, and enhancing benefits to a participant's community. This approach may serve as a model to help researchers, ethicists, and regulators to identify opportunities to better respect and support participants in future research that entails some risk to these participants as well as to improve the quality of research for people with kidney disease.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are common, heterogeneous, and morbid diseases. Mechanistic characterization of CKD and AKI in patients may facilitate a precision medicine approach to prevention, diagnosis, and treatment. The Kidney Precision Medicine Project aims to ethically and safely obtain kidney biopsies from participants with CKD or AKI, create a reference kidney atlas, and characterize disease subgroups to stratify patients based on molecular features of disease, clinical characteristics, and associated outcomes. An additional aim is to identify critical cells, pathways, and targets for novel therapies and preventive strategies. This project is a multicenter prospective cohort study of adults with CKD or AKI who undergo a protocol kidney biopsy for research purposes. This investigation focuses on kidney diseases that are most prevalent and therefore substantially burden the public health, including CKD attributed to diabetes or hypertension and AKI attributed to ischemic and toxic injuries. Reference kidney tissues (for example, living kidney donor biopsies) will also be evaluated. Traditional and digital pathology will be combined with transcriptomic, proteomic, and metabolomics analysis of the kidney tissue as well as deep clinical phenotyping for supervised and unsupervised subgroup analysis and systems biology analysis. Participants will be followed prospectively for ten years to ascertain clinical outcomes. Cell types, locations, and functions will be characterized in health and disease in an open, searchable, online kidney tissue atlas. All data from the Kidney Precision Medicine Project will be made readily available for broad use by scientists, clinicians, and patients.

Rhizosphere bacteria influence the growth of their host plant by consuming and producing metabolites, nutrients, and antibiotic compounds within the root system that affect plant metabolism. Under Fe-limited growth conditions, different plant and microbial species have distinct Fe acquisition strategies, often involving the secretion of strong Fe-binding chelators that scavenge Fe and facilitate uptake., Iron (Fe) availability has well-known effects on plant and microbial metabolism, but its effects on interspecies interactions are poorly understood. The purpose of this study was to investigate metabolite exchange between the grass Brachypodium distachyon strain Bd21 and the soil bacterium Pseudomonas fluorescens SBW25::gfp/lux (SBW25) during Fe limitation under axenic conditions. We compared the transcriptional profiles and root exudate metabolites of B. distachyon plants grown semihydroponically with and without SBW25 inoculation and Fe amendment. Liquid chromatography-mass spectrometry analysis of the hydroponic solution revealed an increase in the abundance of the phytosiderophores mugineic acid and deoxymugineic acid under Fe-limited conditions compared to Fe-replete conditions, indicating greater secretion by roots presumably to facilitate Fe uptake. In SBW25-inoculated roots, expression of genes encoding phytosiderophore biosynthesis and uptake proteins increased compared to that in sterile roots, but external phytosiderophore abundances decreased. P. fluorescens siderophores were not detected in treatments without Fe. Rather, expression of SBW25 genes encoding a porin, a transporter, and a monooxygenase was significantly upregulated in response to Fe deprivation. Collectively, these results suggest that SBW25 consumed root-exuded phytosiderophores in response to Fe deficiency, and we propose target genes that may be involved. SBW25 also altered the expression of root genes encoding defense-related enzymes and regulators, including thionin and cyanogenic glycoside production, chitinase, and peroxidase activity, and transcription factors. Our findings provide insights into the molecular bases for the stress response and metabolite exchange of interacting plants and bacteria under Fe-deficient conditions. IMPORTANCE Rhizosphere bacteria influence the growth of their host plant by consuming and producing metabolites, nutrients, and antibiotic compounds within the root system that affect plant metabolism. Under Fe-limited growth conditions, different plant and microbial species have distinct Fe acquisition strategies, often involving the secretion of strong Fe-binding chelators that scavenge Fe and facilitate uptake. Here, we studied interactions between P. fluorescens SBW25, a plant-colonizing bacterium that produces siderophores with antifungal properties, and B. distachyon, a genetic model for cereal grain and biofuel grasses. Under controlled growth conditions, bacterial siderophore production was inhibited in the root system of Fe-deficient plants, bacterial inoculation altered transcription of genes involved in defense and stress response in the roots of B. distachyon, and SBW25 degraded phytosiderophores secreted by the host plant. These findings provide mechanistic insight into interactions that may play a role in rhizosphere dynamics and plant health in soils with low Fe solubility.

The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell’s physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ. Tissue cytometry is a promising new microscopy technique that can be used to enumerate and characterize each cell in a tissue. Here the authors describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues.

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with isobaric labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of >3,000 proteins and reliable quantification of >2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with isobaric labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.

Water column oxygen (O2)-deficiency shapes food-web structure by progressively directing nutrients and energy away from higher trophic levels into microbial community metabolism resulting in fixed nitrogen loss and greenhouse gas production. Although respiratory O2 consumption during organic matter degradation is a natural outcome of a productive surface ocean, global-warming-induced stratification intensifies this process leading to oxygen minimum zone (OMZ) expansion. Here, we describe useful tools for detection and quantification of potential key microbial players and processes in OMZ community metabolism including quantitative polymerase chain reaction primers targeting Marine Group I Thaumarchaeota, SUP05, Arctic96BD-19, and SAR324 small-subunit ribosomal RNA genes and protein extraction methods from OMZ waters compatible with high-resolution mass spectrometry for profiling microbial community structure and functional dynamics.

Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabolism. These include reactions catalyzed by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and methyl-coenzyme M reductase (MCR). The latter enzyme is central to C-H bond formation and cleavage underlying methanogenic and reverse methanogenic phenotypes. Here, we describe a set of novel tools for the detection and quantification of H4MPT-linked C1 transfer reactions mediated by uncultivated anaerobic methane-oxidizing archaea (ANME). These tools include polymerase chain reaction primers targeting ANME MCR subunit A subgroups and protein extraction methods from marine sediments compatible with high-resolution mass spectrometry for profiling community structure and functional dynamics.

Human platelets play a key role in homeostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 122 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls and 117 proteins whose abundances changed during a 5-day storage period. Notably our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting “high risk platelets” that render platelet transfusion less effective or even unsafe.

Mass spectrometry-based proteomics is a powerful analytical tool for investigating pathogens and their interactions within a host. The sensitivity of such analyses provides broad proteome characterization, but the sample-handling procedures must first be optimized to ensure compatibility with the technique and to maximize the dynamic range of detection. The decision-making process for determining optimal growth conditions, preparation methods, sample analysis methods, and data analysis techniques in our laboratory is discussed herein with consideration of the balance in sensitivity, specificity, and biomass losses during analysis of host-pathogen systems.

Human Vgamma2Vdelta2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vgamma2Vdelta2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vgamma2Vdelta2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vgamma2Vdelta2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vgamma2Vdelta2 T cells in vivo upon engraftment into severe combined immunodeficiency-beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vgamma2Vdelta2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vgamma2Vdelta2 T cells to respond to a broad array of pathogens using this pathway.

A retrospective survey of pediatric patients infected with respiratory pathogens identified increases in glucose metabolism in the lungs of several individuals. After viral infection the host cell mounts an energetic and resource intensive antiviral response, while the virus exploits the host to generate progeny. Using absolute quantification with Host-Influenza Proteomics (HIP) we sought to characterize changes in host cell homeostasis that contribute to these competing processes. Like in the patients, we found gross metabolic changes occurring after influenza infection in primary human respiratory cells as well as dendritic cells. This new metabolic phenotype is distinct from immune activation and involves increases in glycolysis and glutaminolysis, decreased fatty acid β-oxidation concomitant to non-canonical TCA activity. Based on these findings we developed a metabolic drug repurposing screen in human bronchial epithelial cells and high-throughput viral tittering system. We screened over 160 metabolic substrates and small molecules to identify responsive prophylactic and therapeutic pathways (e.g. lipid and glucose metabolism respectively). One compound known to alter glucose sensitivity, NVP-BEZ235, reduced viral titers and significantly increased survival in vivo after lethal influenza challenge. Human PET scans followed by HIP analysis elucidated a unique metabolic signature in influenza-infection exposing targets for therapeutic intervention.

Human gammadelta T cells containing the Vgamma2Vdelta2 (Vgamma9Vdelta2) T cell receptor are stimulated by a broad variety of small, phosphorus-containing antigenic molecules called phosphoantigens. The structures of several species present in both Mycobacteria (TUBags1-4) and in Escherichia coli have been reported to contain a formyl-alkyl diphosphate core. Here we report the synthesis of the lead member of the series, 3-formyl-1-butyl diphosphate. This compound has low activity for gammadelta T cell stimulation, unlike its highly active isomer (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate, necessitating a revision of the structure of TUBag1. Likewise, the structure of the species identified as the pentyl analog (TUBag 2) is revised to 6-phosphogluconate. These results indicate that neither TUBag1 nor the m/e 275 species proposed for TUBag2 are 3-formyl-1-alkyl diphosphates, leading to the conclusion that none of the natural phosphoantigens (TUBags1-4) possess the structures reported previously.

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Airway dendritic cells (DC) initiate the immune response (IR) to respiratory pathogens (e.g.influenza). DC respond by releasing cytokines and presenting antigen to T cells, which activate on encountering the MCHI/peptide complex. This DC-T cell interaction dictates the IR to a given pathogen. Given that antigen presentation couples the innate and adaptive immune responses, a better understanding of these processes is vital to basic immune research and to therapeutic and vaccine development. To elucidate the underlying mechanisms of these processes we elected to identify DC proteins and quantify their changes in abundance using 18O labeling and LTQ-Orbit-trap-MS. Presuming the import of membrane protein dynamics on DCs, we developed a high pressure trypsinization technique to denature and digest insoluble proteins. We identified >1500 DC proteins, 20% of which change in abundance (i.e. ≥ 2 fold) early in infection. Unexpectedly this method detected infection induced changes in membrane proteins exceeding soluble protein dynamics. Analysis of these preliminary data indicates major shifts in DC metabolic and antigen processing and presentation pathways occur as a result of infection. Network analysis resulted in the identification of two critical modulator proteins; one of these was NFκB, a well known modulator of the immune response and another protein previously unidentified as an IR modulator. Together these data revealed new and important aspects of the immune response.