Defining HLA-II Ligand Processing and Binding Rules with Mass Spectrometry Enhances Cancer Epitope Prediction.

Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.

Interpreting metabolic complexity via isotope-assisted metabolic flux analysis.

Isotope-assisted metabolic flux analysis (iMFA) is a powerful method to mathematically determine the metabolic fluxome from experimental isotope labeling data and a metabolic network model. While iMFA was originally developed for industrial biotechnological applications, it is increasingly used to analyze eukaryotic cell metabolism in physiological and pathological states. In this review, we explain how iMFA estimates the intracellular fluxome, including data and network model (inputs), the optimization-based data fitting (process), and the flux map (output). We then describe how iMFA enables analysis of metabolic complexities and discovery of metabolic pathways. Our goal is to expand the use of iMFA in metabolism research, which is essential to maximizing the impact of metabolic experiments and continuing to advance iMFA and biocomputational techniques.

Metabolic Diversity in Human Non-Small Cell Lung Cancer Cells.

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture.

Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive 13C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins. CHO cells were cultured in media formulations missing a single amino acid or glucose supplemented with a 13C-tracer of the missing substrate, followed by gas chromatography-mass spectrometry (GC-MS) analysis to track labeled carbon flows and identify by-products. We tracked the sources of all secreted by-products and verified the identity of 45 by-products, majority of which were derived from glucose, leucine, isoleucine, valine, tyrosine, tryptophan, methionine, and phenylalanine. In addition to by-products identified previously, we identified several metabolites including 2-hydroxyisovaleric acid, 2-aminobutyric acid, L-alloisoleucine, ketoisoleucine, 2-hydroxy-3-methylvaleric acid, desmeninol, and 2-aminobutyric acid. When added to CHO cell cultures at different concentrations, certain metabolites inhibited cell growth while others including 2-hydroxy acids, surprisingly, reduced lactate accumulation. In vitro enzymatic analysis indicated that 2-hydroxy acids were metabolized by lactate dehydrogenase suggesting a possible mechanism for lowered lactate accumulation, e.g., competitive substrate inhibition. The 13C-labeling assisted metabolomics pipeline developed and the metabolites identified will serve as a springboard to reduce undesirable by-products accumulation and alleviate inefficient substrate utilization in mammalian cultures used for biomanufacturing and other applications through altered media formulations and pathway engineering strategies.

Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Epigenetic MRI: Noninvasive imaging of DNA methylation in the brain.

Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here, we present a method, epigenetic MRI (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene-expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C-labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.

The key role of glutamine for protein expression and isotopic labeling in insect cells.

Nuclear magnetic resonance studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotope-labeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N nuclear magnetic resonance spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the tricarboxylic acid cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-α,ß,ß-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled ß1-adrenergic receptor using the synthesized 15N2-glutamine-α,ß,ß-d3.

Spatio-Temporal Study of Galactolipid Biosynthesis in Duckweed Using Mass Spectrometry Imaging and in vivo Isotope Labeling.

Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism at cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatio-temporal dynamics of lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization-MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over a 15-day time period. When they were partially labeled after 5 d, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain and the entire molecule. The temporal change in the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their mass spectrometry images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues and the newly grown daughter fronds. Besides, two additional labeling experiments, (i) 13CO2 labeling and (ii) backward labeling of completely 50% D2O-labeled L. minor in H2O media, confirm the observations in forward labeling. Furthermore, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling using MSI to provide spatio-temporal details in lipid biosynthesis in plant development.

Uniform [13C,15N]-labeled and glycosylated IgG1 Fc expressed in Saccharomyces cerevisiae.

Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [13C]-glucose, and [15N]-ammonium chloride with complexity comparable to E. coli. Here we report two vectors for expressing the crystallizable fragment (Fc) of IgG1 for secretion into the culture medium, utilizing the ADH2 or DDI2 promoters. We also report a strategy to optimize the expression yield using orthogonal Taguchi arrays. Lastly, we developed two different media formulations, a standard medium which provides 86-92% 15N and 30% 13C incorporation into the polypeptide, or a rich medium which provides 98% 15N and 95% 13C incorporation as determined by mass spectrometry. This advance represents an expression and optimization strategy accessible to experimenters with the capability to grow and produce proteins for NMR-based experiments using E. coli.

Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns.

We present an economic and straightforward method to introduce 13C-19F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13C, 2,3,5,6-2H4] 4-fluorophenylalanine from the commercially available isotope sources [2-13C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.

Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C', 13Cα, 13Cß resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR.

Heterostructured Co-Doped-Cu2O/Cu Synergistically Promotes Water Dissociation for Improved Electrochemical Nitrate Reduction to Ammonia.

Electrochemical nitrate reduction reaction (NO3RR) has recently emerged as a promising approach for sustainable ammonia synthesis and wastewater treatment, while the activity and selectivity for ammonia production have remained low. Herein, rational design and controllable synthesis of heterostructured Co-doped Cu2O/Cu nanoparticles embedded in carbon framework (Co-Cu2O/Cu@C) is reported for NO3RR. The Co-Cu2O/Cu@C exhibits a high ammonia yield rate of 37.86 mg h-1 mg-1 cat. with 98.1% Faraday efficiency, which is higher than those obtained for most of the Cu-based catalysts under similar conditions. Density functional theory calculations indicated that the strong electronic interactions at Cu/Co-Cu2O interface facilitate the N species deoxygenation process and doping of Co promotes water dissociation to generate *H for the N species hydrogenation process, leading to enhanced NO3RR performance. This work provides a new design strategy toward high-performance catalysts toward NO3RR for ammonia generation.

Synthesis of a 13C/2H Labeled Building Block to Probe the Phosphotyrosine Interactome Using Biomolecular NMR Spectroscopy.

Phosphotyrosine (pTyr) recognition coordinates the assembly of protein complexes, thus controlling key events of cell cycle, cell development and programmed cell death. Although many aspects of membrane receptor function and intracellular signal transduction have been deciphered in the last decades, the details of how phosphorylation alters protein-protein interaction and creates regulating switches of protein activity and localization often remains unclear. We developed a synthetic route to a protected phophotyrosine building block with isolated 13C-1H spins in the aromatic ring. The compound can be used for solid phase peptide synthesis (SPPS) and readily applied to study affinity, dynamics and interactions on an atomic level using NMR spectroscopy. As a first example, we prepared an isotopologue of a pTyr containing 12mer peptide (pY1021) as part of the platelet-derived growth factor to analyze the binding to the phospholipase C-γ (PLCγ-1) SH2 domain.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

One-shot 13 C15 N-metabolic flux analysis for simultaneous quantification of carbon and nitrogen flux.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13 C15 N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13 C15 N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

Chemical isotope labeling for quantitative proteomics.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

Chemical derivatization strategy for mass spectrometry-based lipidomics.

Lipids, serving as the structural components of cellular membranes, energy storage, and signaling molecules, play the essential and multiple roles in biological functions of mammals. Mass spectrometry (MS) is widely accepted as the first choice for lipid analysis, offering good performance in sensitivity, accuracy, and structural characterization. However, the untargeted qualitative profiling and absolute quantitation of lipids are still challenged by great structural diversity and high structural similarity. In recent decade, chemical derivatization mainly targeting carboxyl group and carbon-carbon double bond of lipids have been developed for lipidomic analysis with diverse advantages: (i) offering more characteristic structural information; (ii) improving the analytical performance, including chromatographic separation and MS sensitivity; (iii) providing one-to-one chemical isotope labeling internal standards based on the isotope derivatization regent in quantitative analysis. Moreover, the chemical derivatization strategy has shown great potential in combination with ion mobility mass spectrometry and ambient mass spectrometry. Herein, we summarized the current states and advances in chemical derivatization-assisted MS techniques for lipidomic analysis, and their strengths and challenges are also given. In summary, the chemical derivatization-based lipidomic approach has become a promising and reliable technique for the analysis of lipidome in complex biological samples.

Size matters: Aerobic methane oxidation in sediments of shallow thermokarst lakes.

Shallow thermokarst lakes are important sources of greenhouse gases (GHGs) such as methane (CH4 ) and carbon dioxide (CO2 ) resulting from continuous permafrost thawing due to global warming. Concentrations of GHGs dissolved in water typically increase with decreasing lake size due to coastal abrasion and organic matter delivery. We hypothesized that (i) CH4 oxidation depends on the natural oxygenation gradient in the lake water and sediments and increases with lake size because of stronger wind-induced water mixing; (ii) CO2 production increases with decreasing lake size, following the dissolved organic matter gradient; and (iii) both processes are more intensive in the upper than deeper sediments due to the in situ gradients of oxygen (O2 ) and bioavailable carbon. We estimated aerobic CH4 oxidation potentials and CO2 production based on the injection of 13 C-labeled CH4 in the 0-10 cm and 10-20 cm sediment depths of small (~300 m2 ), medium (~3000 m2 ), and large (~106 m2 ) shallow thermokarst lakes in the West Siberian Lowland. The CO2 production was 1.4-3.5 times stronger in the upper sediments than in the 10-20 cm depth and increased from large (158 ± 18 nmol CO2 g-1 sediment d.w. h-1 ) to medium and small (192 ± 17 nmol CO2 g-1 h-1 ) lakes. Methane oxidation in the upper sediments was similar in all lakes, while at depth, large lakes had 14- and 74-fold faster oxidation rates (5.1 ± 0.5 nmol CH4 -derived CO2 g-1 h-1 ) than small and medium lakes, respectively. This was attributed to the higher O2 concentration in large lakes due to the more intense wind-induced water turbulence and mixing than in smaller lakes. From a global perspective, the CH4 oxidation potential confirms the key role of thermokarst lakes as an important hotspot for GHG emissions, which increase with the decreasing lake size.

Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using 15N-labeled crystallin standards.

Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, ßA2, ßA3/A1, ßA4, ßB1, ßB2, ßB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the 15N artificial lens standard. Proteins were trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data independent MS2 scans, and data analysis performed using Skyline software. Crystallin abundances were measured in both human donor lenses and in transgenic mouse αA N101D cataract lenses. Technical replicates of human crystallin abundance measurements were performed with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 4.5/1 to 2/1. γS-crystallin also increased nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while ßB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins ßA2, ßB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. The high content of αA and the transient abundance of ßA2, ßB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. ßB1 becoming the most abundant crystallin may result from its role in promoting higher order ß-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for future crystallin studies. 15N labeling of these crystallins will be useful to accurately quantify crystallins in lens anatomic regions, as well as measure the composition of insoluble light scattering crystallin aggregates. The standards will also be useful to measure the abundance of crystallins expressed in transgenic animal models.