Isotope Labeling Research Articles

Region-specific mouse brain ganglioside distribution revealed by an improved isobaric aminoxyTMT labeling strategy with automated data processing.

Gangliosides are specialized glycosphingolipids most abundant in the central nervous system. Their complex amphiphilic structure is essential to the formation of membrane lipid rafts and for molecular recognition. Dysfunction of lipid rafts and ganglioside metabolism has been linked to cancer, metabolic disorders, and neurodegenerative disorders. Changes in ganglioside concentration and diversity during the progression of disease have made them potential biomarkers for early detection and shed light on disease mechanisms. Chemical derivatization facilitates whole ion analysis of gangliosides while improving ionization, providing rich fragmentation spectra, and enabling multiplexed analysis schemes such as stable isotope labeling. In this work, we report improvement to our previously reported isobaric labeling methodology for ganglioside analysis by increasing buffer concentration and removing solid-phase extraction desalting for a more complete and quantitative reaction. Identification and quantification of gangliosides are automated through MS-DIAL with an in-house ganglioside derivatives library. We have applied the updated methodology to relative quantification of gangliosides in six mouse brain regions (cerebellum, pons/medulla, midbrain, thalamus/hypothalamus, cortex, and basal ganglia) with 2 mg tissue per sample, and region-specific distributions of 88 ganglioside molecular species are described with ceramide isomers resolved. This method is promising for application to comparative analysis of gangliosides in biological samples.

Aspirin reprogrammes colorectal cancer cell metabolism and sensitises to glutaminase inhibition

BackgroundTo support proliferation and survival within a challenging microenvironment, cancer cells must reprogramme their metabolism. As such, targeting cancer cell metabolism is a promising therapeutic avenue. However, identifying tractable nodes of metabolic vulnerability in cancer cells is challenging due to their metabolic plasticity. Identification of effective treatment combinations to counter this is an active area of research. Aspirin has a well-established role in cancer prevention, particularly in colorectal cancer (CRC), although the mechanisms are not fully understood.MethodsWe generated a model to investigate the impact of long-term (52 weeks) aspirin exposure on CRC cells, which has allowed us comprehensively characterise the metabolic impact of long-term aspirin exposure (2–4mM for 52 weeks) using proteomics, Seahorse Extracellular Flux Analysis and Stable Isotope Labelling (SIL). Using this information, we were able to identify nodes of metabolic vulnerability for further targeting, investigating the impact of combining aspirin with metabolic inhibitors in vitro and in vivo.ResultsWe show that aspirin regulates several enzymes and transporters of central carbon metabolism and results in a reduction in glutaminolysis and a concomitant increase in glucose metabolism, demonstrating reprogramming of nutrient utilisation. We show that aspirin causes likely compensatory changes that render the cells sensitive to the glutaminase 1 (GLS1) inhibitor—CB-839. Of note given the clinical interest, treatment with CB-839 alone had little effect on CRC cell growth or survival. However, in combination with aspirin, CB-839 inhibited CRC cell proliferation and induced apoptosis in vitro and, importantly, reduced crypt proliferation in Apcfl/fl mice in vivo.ConclusionsTogether, these results show that aspirin leads to significant metabolic reprogramming in colorectal cancer cells and raises the possibility that aspirin could significantly increase the efficacy of metabolic cancer therapies in CRC.

Nitrogen fixation and transcriptome of a new diazotrophic Geomonas from paddy soils.

The ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.

Biosynthesis of Phomactin Platelet Activating Factor AntagonistRequires a Two-Enzyme Cascade.

Phomactin diterpenoids possess a unique bicyclo[9.3.1]-pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from amarine fungus,Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate-analogue FGGPP and elucidated the novel metal ion binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This work provides the first insights into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Biosynthesis of Phomactin Platelet Activating Factor Antagonist Requires a Two‐Enzyme Cascade

AbstractPhomactin diterpenoids possess a unique bicyclo[9.3.1]pentadecane skeleton with multiple oxidative modifications, and are good platelet‐activating factor (PAF) antagonists that can inhibit PAF‐induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from a marine fungus, Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate analogue FGGPP and elucidated the novel metal‐ion‐binding mode and enzymatic mechanism of PhmA through site‐directed mutagenesis. This study provides the first insight into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Nickel‐Catalyzed Isotopic Labeling: Synthesis of Oxygen‐18‐Labeled Esters from Amides

AbstractA method is developed for preparing 18O‐labeled esters through a Ni‐catalyzed three‐component reaction of amides, alkyl halides, and 18O‐labeled water. This method demonstrated excellent selectivity and compatibility with various amides and alkyl halides, allowing the synthesis of diverse isotopically labelled esters. Ni‐catalyzed reaction of alkyl bromides, carboxylates that generated from amides, and water in the presence of a base formed the desired esters.

Detection and Characterization of Active Microbial Cells in Salt Cavern Brine

Salt caverns have been used for decades as natural gas storage facilities and are now target of large-scale underground H2 storage to secure national energy transition goals. Contrary to CH4, H2 is a universal electron donor for microbial anaerobic respiration. Suitable electron acceptors are sulfate and carbonate, which dissolve from gypsum, anhydrite and lime that can make up 10 % of subsurface salt formations. Whilst sulfate reduction is inherently linked to the formation of H2S, microbial CO2 reduction can generate acetate, which can be used as carbon source by diverse microorganisms. Thus, supporting other microbial side effects, such as H2S formation, clogging and H2 consumption. However, microbial diversity and activity in salt caverns are selectively controlled by salt concentrations close to saturation and limited availability of organic carbon. If these conditions allow for microbial activity was investigated in our study. To circumvent long enrichment times associated with high salinity and limited nutrient availability, we used a stable isotope labelling approach combined with nano-scale secondary ion mass spectrometry analysis (SIP-nanoSIMS) to investigate H2-dependant microbial activity in two brine samples and compared them with that of an extremely halophilic enrichment culture (MP-32). Heavy carbonate and water (13CO2 and 2H2O) served as tracers for microbial activity. Microbial H2 consumption was additionally investigated in microcosm experiments with brine and rock salt over a period of 200 days. Setups with MP-32 served as a positive control. Subsequently, MP-32 was selected for metagenome sequencing to explore potential metabolic pathways and strategies for osmoadaptation. Analysis of the microbial community composition in brine revealed that members of the Desulfohalobiaceae, Halobacteria and Halanaerobiales were present in all caverns and their relative abundance increased during incubation with H2 as electron donor although sulfate reduction was not observed. But incubation with H2 resulted in an increased uptake of 13C from 13CO2 in 1.6 to 3.6 % of the cells compared to incubations without H2. Uptake of 2H from 2H2O was detected in 20 to 30 % of the cells and was higher when H2 was not offered as an electron donor. Similar results were obtained from the enrichment culture MP-32, which was grown in medium with reduced salinity compared to the salt cavern brine. Uptake of 13C was 10-fold higher when incubated with H2 and nearly all cells incorporated 2H with and without H2. A total of eight metagenome-assembled genomes (MAGs) with a completion of more than 90 % could be recovered from MP-32. Two of them belonged to Desulfohalobiaceae and can be characterized as autotrophic sulfate reducers by means of the Acetyl-Coenzyme A pathway that compensate osmotic stress by synthesizing small organic molecules. Collectively, our findings provide a new approach to study microbial activity that is strongly impacted by high salinity and an improved understanding of their genomic potential.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics.

The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Uncovering the Dinuclear Mechanism of NO2-Involved NH3-SCR over Supported V2O5/TiO2 Catalysts.

Commercial vanadium oxide catalysts exhibit high efficiency for the selective catalytic reduction (SCR) of NO with NH3, especially in the presence of NO2 (i.e., occurrence of fast NH3-SCR). The high-activity sites and their working principle for the fast NH3-SCR reaction, however, remain elusive. Here, by combining in situ spectroscopy, isotopic labeling experiments, and density functional theory (DFT) calculations, we demonstrate that polymeric vanadyl species act as the main active sites in the fast SCR reaction because the coupling effect of the polymeric structure alters the elementary reaction step and effectively avoids the high energy barrier of the rate-determining step over monomeric vanadyl species. This study unveils the high-activity dinuclear mechanism of the NO2-involved SCR reaction over vanadia-based catalysts and provides a fundamental basis for developing high-efficiency and low V2O5-loading SCR catalysts.

Mechanism for Generating H2 O2 at Water-Solid Interface by Contact-Electrification.

The recent intensification of the study of contact-electrification at water-solid interfaces and its role in physicochemical processes lead to the realization that electron transfers during water-solid contact-electrification can drive chemical reactions. This mechanism, named contact-electro-catalysis (CEC), allows chemically inert fluorinated polymers to act like single electrode electrochemical systems. This study shows hydrogen peroxide (H2 O2 ) is generated from air and deionized water, by ultrasound driven CEC, using fluorinated ethylene propylene (FEP) as the catalyst. For a mass ratio of catalyst to solution of 1:10000, at 20°C, the kinetic rate of H2 O2 evolution reaches 58.87mmolL-1 gcat -1 h-1 . Electron paramagnetic resonance (EPR) shows electrons are emitted in the solution by the charged FEP, during ultrasonication. EPR and isotope labelling experiments show H2 O2 is formed from hydroxyl radicals (HO• ) or two superoxide radicals (O2 •- ) generated by CEC. Finally, it is traditionally believed such radicals migrate in the solution by Brownian diffusion prior to reactions. However, ab-initio molecular dynamic calculations reveal the radicals can react by exchanging protons and electrons through the hydrogen bonds network of water, i.e., owing to the Grotthuss mechanism. This mechanism can be relevant to other systems, artificial or natural, generating H2 O2 from air and water.

Microbial activities along a 20 million-year-old pristine oil reservoir

Studies on oil reservoir microbiology typically take samples from producing reservoirs and sample fluids that have been pumped to the surface. This comes with problems since producing oil reservoirs are affected by production processes leading to changes in environmental conditions and the natural microbiome. Hence, pumped samples do not display an unaltered picture of the spatial distribution and composition of the microorganisms in the reservoir. We took 13 samples from a freshly drilled sediment core of a pristine, heavily biodegraded oil reservoir in the North Sea. Core samples originated from above, within, and below the reservoir. 16S rRNA gene amplicon sequencing of the microbiome revealed distinct differences between sediments and formation water, indicating that studies on microbiomes from formation water alone are not necessarily representative for the microbial processes in an oil reservoir. Fluorescence microscopy showed that microorganisms live in small microcolonies on the sediment surface. CT-scanning with image analysis visualized the water phase distribution inside the reservoir sediments and clearly indicated water-filled voids that might be habitable for microorganisms, enlarging the surface for potential biodegradation. Employing microcosm experiments and reverse isotope labelling, we were able to determine the first degradation rates measured from cores above, within, and below a reservoir ranging from no activity up to 1 mM CO2/(gsediment x year), Results indicate significant degradation potential from autochthonous microorganisms in the reservoir above the water-contact-zone. Evading the general issues of produced oil samples for studying microbiomes results in a more realistic picture of an oil reservoir unaffected by production artefacts.

Protein methylation characterization using NMR without isotopic labeling

Protein methylation is crucial in epigenetics, and targeting the involved methyltransferases shows great potential for therapeutic intervention with several inhibitors in clinical trials for oncology indications. Therefore, characterization of protein methylation is essential for understanding the methyltransferase function and discovering chemical inhibitors and antagonists. While NMR has been used to measure methylation rates, isotopic labeling of protein or methyl donors can be costly and cannot characterize demethylation of proteins extracted from natural sources. Our method employs a four-quantum filter 1H-13C experiment that selectively detects methyl groups, providing a simple way to characterize methylation and demethylation features of methyltransferases and demethylases, respectively, without requiring isotopic labeling. In our experiments, we successfully observed the methylation of H3 under lysate from various cells and tissues of mice with cancerous growth. The results revealed that H3 undergoes both mono- and dimethylation in all the tested lysates, but at varying rates and degrees. Significantly lower H3 methylation rates and levels were observed in both cervical tumor and breast tumor lysates compared with the corresponding cancerous cells and healthy cells lysates. These findings highlight the variability of histone H3 methylation patterns among healthy cells, cancerous cells, tumor tissues, and different tumor types, and suggest that this method has great potential in facilitating the development of effective interventions against these diseases. By characterizing the methylation features of suspected tumors or areas of concern, it provides valuable insights into the underlying mechanisms of cancer development and aids in identifying potential targets for therapeutic interventions.

Label-Free Quantification of Digital Nanorods Assembled from Discrete Oligourethane Amphiphiles.

Polymeric nanoparticles (NPs) have been extensively designed for theranostic agent delivery. Previous methods for tracking their biological behavior and assessing theranostic efficacy heavily rely on fluorescence or isotope labeling. However, these labeling techniques may alter the physicochemical properties of the labeled NPs, leading to inaccurate biodistribution information. Therefore, it is highly desirable to develop label-free techniques for accurately assessing the biological fate of polymeric NPs. Here, we create discrete oligourethane amphiphiles (DOAs) with methoxy (OMe), hydroxyl (OH), and maleimide (MI) moieties at the dendritic oligo(ethylene glycol) (dOEG) ends. We obtained four types of digital nanorods (NRs) with distinct surface functional groups through self-assembly of a single DOA (OMe and OH NRs) or coassembly of two DOAs (OMe-MI and OH-MI NRs). These unique NRs can be directly quantified in a label-free manner by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Specifically, OMe-MI NRs exhibited the best blood circulation, and OH-MI showed the highest area under the curve (AUC) value after intravenous injection. Biodistribution studies demonstrated that MI-containing NRs generally had lower accumulation in the liver and spleen compared to that of MI-free NRs, except for the comparison between OMe and OMe-MI NRs in the liver. Proteomics studies unveiled the formation of distinct protein coronas that may greatly affect the biological behavior of NRs. This study not only provides a label-free technique for quantifying the pharmacokinetics and biodistribution of polymeric NRs but also highlights the significant impact of surface functional groups on the biological fate of polymeric NPs.

Preparation and Spectroscopic Identification of the Cyclic CO2 Dimer 1,2-Dioxetanedione.

We report the preparation and infrared spectroscopic identification of 1,2-dioxetanedione, which is one of the two possible cyclic dimers of carbon dioxide. We prepared this hitherto experimentally incompletely characterized species in a solid nitrogen matrix at 3 K from the reaction of oxalyl dichloride with the urea·hydrogen peroxide complex. Surprisingly, irradiation at 254 nm does not lead to its dissociation into carbon dioxide but rather yields cyclic carbon trioxide. We further assert our spectroscopic assignments by 18O isotopic labeling and high-level N-electron valence state perturbation theory and coupled-cluster computations. The successful isolation of 1,2-dioxetanedione supports its viability as the postulated high-energy intermediate in the well-known and ubiquitously exploited "peroxyoxalate" chemiluminescent system.

Generation mechanism and emission characteristics of N2O and NOx in ammonia-diesel dual-fuel engine

Ammonia, as a hydrogen-derived fuel, primarily emits N2O and NOx when burned in an engine. The emissions originate from ammonia fuel and nitrogen in the air. In this paper, the N of N2 was labeled by isotope labeling method, so that the nitrogen element in the formation path of the thermal N2O and NOx was labeled, and the chemical reaction path of the formation and evolution of the fuel and thermal nitrogen oxide was defined. In addition, the spatiotemporal distribution characteristics, generation and evolution rules of fuel and thermal nitrogen oxide in ammonia-diesel dual-fuel engine, as well as the influence of different ammonia energy ratios on the generation ratio of two types of N2O and NOx were studied by combining experiment and simulation. The ammonia energy ratio is defined as the proportion of energy provided by ammonia in a constant total lower calorific value. The results showed that when the ammonia energy ratio increased from 20 % to 60 %, the emissions of NO and NO2 decreased, but the proportion of fuel NO and NO2 increased. The proportion of N2O in nitrogen oxide emission increased from 7.3 % to 20 %. Fuel N2O accounts for about 90 % of N2O emission. Fuel N2O and NOx were mainly generated in ammonia-rich, low-temperature regions and thermal N2O and NOx were mainly generated in the high-temperature regions. Temperature significantly affected N2O generation.

Comprehensive quantification of metabolic flux during acute cold stress in mice

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

Effects of Solvent Evaporation Methods and Short-Term Room Temperature Storage on High-Coverage Cellular Metabolome Analysis.

Cellular metabolomics provides insights into the metabolic processes occurring within cells and can help researchers understand how these processes are regulated and how they relate to cellular function, health, and disease. In this technical note, we investigated the effects of solvent evaporation equipment and storage condition on high-coverage cellular metabolomics. We previously introduced a robust CIL LC-MS-based cellular metabolomics workflow that encompasses various steps, including cell harvest, metabolic quenching, cell lysis, metabolite extraction, differential chemical isotope labeling, and LC-MS analysis. This workflow has consistently served as the cornerstone of our collaborative research and service projects. As a core facility catering to users with diverse research needs and financial resources, we have encountered scenarios requiring short-term sample storage. For example, the need often arises to transport samples at room temperature from user sites to our core facility. Herein, we present a study in which we compared different solvent evaporation methods (specifically, the nitrogen blowdown evaporator, SpeedVac concentrator, and lyophilizer) and diverse storage conditions (including dried samples stored in a freezer, samples stored in a freezer with methanol, dried samples stored at room temperature, and samples stored at room temperature with methanol). Our findings indicate that the choice of solvent evaporation equipment did not significantly impact the cellular metabolome. However, we observed a noteworthy change in the metabolome after 7 days of storage when cells were stored with methanol, regardless of whether they were kept at -80 °C or room temperature, in contrast to cells that were dried and frozen. Importantly, we detected no significant alterations in cells that were dried and stored at room temperature. In conclusion, to ensure the production of high-quality CIL LC-MS metabolomics results, we strongly recommend that, in situations where low-temperature storage is not feasible, cell samples should be thoroughly dried before storage or shipment at room temperature.

Cysteine Counting via Isotopic Chemical Labeling for Intact Mass Proteoform Identifications in Tissue.

Top-down proteomics, the tandem mass spectrometric analysis of intact proteoforms, is the dominant method for proteoform characterization in complex mixtures. While this strategy produces detailed molecular information, it also requires extensive instrument time per mass spectrum obtained and thus compromises the depth of proteoform coverage that is accessible on liquid chromatography time scales. Such a top-down analysis is necessary for making original proteoform identifications, but once a proteoform has been confidently identified, the extensive characterization it provides may no longer be required for a subsequent identification of the same proteoform. We present a strategy to identify proteoforms in tissue samples on the basis of the combination of an intact mass determination with a measured count of the number of cysteine residues present in each proteoform. We developed and characterized a cysteine tagging chemistry suitable for the efficient and specific labeling of cysteine residues within intact proteoforms and for providing a count of the cysteine amino acids present. On simple protein mixtures, the tagging chemistry yields greater than 98% labeling of all cysteine residues, with a labeling specificity of greater than 95%. Similar results are observed on more complex samples. In a proof-of-principle study, proteoforms present in a human prostate tumor biopsy were characterized. Observed proteoforms, each characterized by an intact mass and a cysteine count, were grouped into proteoform families (groups of proteoforms originating from the same gene). We observed 2190 unique experimental proteoforms, 703 of which were grouped into 275 proteoform families.