Complementary Mass Spectrometry Research Articles

Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry.

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates, such as fatty acids, carbohydrates, lipids, and amino acids, to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities, which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection: direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF-MS/MS). Using DI-FTICR MS, 9644 metabolic features were detected where 7156 were assigned a molecular formula and 1107 were annotated by accurate mass assignment. Using LC-Q-TOF-MS/MS, 21,428 metabolic features were detected where 285 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 1340 heart metabolites were identified in this study, which span a wide range of polarities including polar (benzenoids, carbohydrates, and nucleosides) as well as nonpolar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results from this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.

EPCO-13. CNMF-SNS, A FRAMEWORK FOR UNSUPERVISED INTEGRATION OF MULTIOMICS DATA, IDENTIFIES INVASION AND RECURRENCE ASSOCIATED PROGRAMS IN GBM

Abstract Glioblastoma (GBM) is an aggressive cancer of the brain for which novel therapies are urgently needed. Heterogeneity in GBM underlies tumor evolution and resistance to therapy and involves genetic diversity of tumor cells, spatially distinct niches harbouring stem-like cells, and immune cells spanning anti- and pro-tumor states. No single profiling methodology can capture all these facets of tumor biology in the same space and time, necessitating multi-omics data collection and computational methods suited for multi-omic integration across and within cohorts. We therefore set out to develop a generalizable framework for integration that can bridge across cohorts and data types. Using consensus non-negative matrix factorization (cNMF), we discover gene expression programs (GEPs) corresponding to cell types and/or states in an unsupervised fashion. Rather than choosing a single rank (number of programs), we identify both high and low-resolution programs for each dataset, and then integrate programs from all datasets and ranks into a solution network space (SNS) on which graph algorithms identify communities of highly similar GEPs identified in all or some datasets. These communities are characterized by gene set enrichment analyses and association with sample metadata. SNS communities can be used to transfer sample metadata across datasets, enabling multi-omics and multi-cohort integration, even in the absence of shared samples or cells. We showcase the utility of cNMF-SNS in integrating multiomics datasets of GBM from single-cell to bulk RNA-Seq and mass spectrometry proteomics. We further demonstrate the power of cNMF-SNS to define and cross-annotate programs identified in 5 complementary mass spectrometry datasets of GBM, layering on survival, driver gene mutation status, clinical features, and imaging features. We demonstrate robust mapping of biologically and clinically relevant processes, highlighting how programs for tumor cell invasion and therapy resistance can be mined for novel therapeutic targets. cNMF-SNS is available at https://github.com/MorrissyLab/cNMF-SNS.

Assessing biodegradation of roadway particles via complementary mass spectrometry and NMR analyses

Roadway particles (RP) that can be collected with on-vehicle system, consist of a mixture of Tire and road wear particles (TRWP) with other traffic-derived particles (exhaust or non-exhaust) and/or biogenic compounds and represent a significant source of xenobiotics, susceptible to reach the different environmental compartments. The study of the RP fate is thus a major challenge to tackle in order to understand their degradation and impact. They offer a variety of carbon sources potentially usable by microorganisms, ranging from the tire-derived plasticizers, vulcanizing agents, protective agents and their transformation products, to other traffic, road and environmental-derived contaminants. A multi-analytical approach was implemented to characterize RP and study their biodegradation. Kinetics of RP extractions were monitored during 21 days in water, methanol, acetone and chloroform to identify leaching and extractable compounds and monitor the particle composition. The results confirmed that hundreds of readily leachable chemicals can be extracted from RP directly into water according to a dynamic process with time while additional poorly soluble compounds remain in the particles. Mass spectrometry (LC-HRMS and GC–MS) allowed us to propose 296 putative compounds using an extensive rubber database. The capacity of 6 bacterial strains, belonging to Rhodococcus, Pseudomonas and Streptomyces genera, to biodegrade RP was then evaluated over 14 days of incubation. The selected strains were able to grow on RP using various substrates. Elastomer monitoring by 1H NMR revealed a significant 12 % decrease of the extractable SBR fraction when the particles were incubated with Rhodococcus ruber. After incubation, the biodegradation of 171 compounds among leachable and extractable compounds was evaluated. Fatty acids and alkanes from rubber plasticizers and paraffin waxes were the most degraded putative compounds by the six strains tested, reaching 75 % of biodegradation for some of them.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53’s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53’s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

High-throughput profiling of RNA modifications by ultra-performance liquid chromatography coupled to complementary mass spectrometry: Methods, quality control, and applications

Although next-generation sequencing technology has been used to delineate RNA modifications in recent years, the paucity of appropriate converting reactions or specific antibodies impedes the accurate characterization and quantification of numerous RNA modifications, especially when these modifications demonstrate wide variations across developmental stages and cell types. In this study, we developed a high-throughput analytical platform coupling ultra-performance liquid chromatograph (UPLC) with complementary mass spectrometry (MS) to identify and quantify RNA modifications in both synthetic and biological samples. Sixty-four types of RNA modifications, including positional isomers and hypermodified ribonucleosides, were successfully monitored within a 16-min single run of UPLC–MS. Two independent methods to cross-validate the purity of RNA extracted from Caenorhabditis elegans (C. elegans) were developed using the coexisting C. elegans and Escherichia coli (E. coli) as a surveillance system. To test the validity of the method, we investigated the RNA modification landscape of three model organisms, C. elegans, E. coli, and Arabidopsis thaliana (A. thaliana). Both the identity and molarity of modified ribonucleosides markedly varied among the species. Moreover, our platform is not only useful for exploring the dynamics of RNA modifications in response to environmental cues (e.g., cold shock) but can also help with the identification of RNA-modifying enzymes in genetic studies. Cumulatively, our method presents a novel platform for the comprehensive analysis of RNA modifications, which will be of benefit to both analytical chemists involved in biomarker discovery and biologists conducting functional studies of RNA modifications.

Unraveling the Bombus terrestris Hemolymph, an Indicator of the Immune Response to Microbial Infections, through Complementary Mass Spectrometry Approaches

Pollinators, including Bombus terrestris, are crucial for maintaining biodiversity in ecosystems and for agriculture. Deciphering their immune response under stress conditions is a key issue for protecting these populations. To assess this metric, we analyzed the B. terrestris hemolymph as an indicator of their immune status. Hemolymph analysis was carried out using mass spectrometry, MALDI molecular mass fingerprinting was used for its effectiveness in assessing the immune status, and high-resolution mass spectrometry was used to measure the impact of experimental bacterial infections on the "hemoproteome". By infecting with three different types of bacteria, we observed that B. terrestris reacts in a specific way to bacterial attacks. Indeed, bacteria impact survival and stimulate an immune response in infected individuals, visible through changes in the molecular composition of their hemolymph. The characterization and label-free quantification of proteins involved in specific signaling pathways in bumble bees by bottom-up proteomics revealed differences in protein expression between the non-experimentally infected and the infected bees. Our results highlight the alteration of pathways involved in immune and defense reactions, stress, and energetic metabolism. Lastly, we developed molecular signatures reflecting the health status of B. terrestris to pave the way for diagnosis/prognosis tools in response to environmental stress.

Proteomic analysis of proteins released by Tritrichomonas foetus: Identification of potential targets for the development of new diagnostic methods

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT.

Synthesis of Perdeuterated Linoleic Acid‐d31 and Chain Deuterated 1‐Palmitoyl‐2‐linoleoyl‐sn‐glycero‐3‐phosphocholine‐d62

AbstractHerein, we report a gram‐scale synthesis of perdeuterated linoleic acid‐d31. The starting materials for the synthesis are two saturated fatty acids, azelaic acid‐d14 and pentanoic acid‐d9, which can be obtained by metal catalysed hydrothermal hydrogen‐deuterium exchange. The synthesis utilises the fatty acids directly via decarboxylative coupling. Copper catalysed coupling of a terminal alkyne intermediate with a propargyl bromide derivative affords a skipped diyne, which can be reduced using P‐2 nickel to obtain the desired cis,cis‐diene geometry. The subsequent synthesis of the tail‐deuterated phospholipid, 1‐palmitoyl‐2‐linoleoyl‐sn‐glycero‐3‐phosphocholine‐d62 (PLPC‐d62) is also described. Optimised reaction conditions were developed to access this phospholipid and its regioisomeric purity was characterised by two complementary mass spectrometry techniques.magnified image

Artificially Intelligent Nanoarray Detects Various Cancers by Liquid Biopsy of Volatile Markers.

Cancer is usually not symptomatic in its early stages. However, early detection can vastly improve prognosis. Liquid biopsy holds great promise for early detection, although it still suffers from many disadvantages, mainly searching for specific cancer biomarkers. Here, a new approach for liquid biopsies is proposed, based on volatile organic compound (VOC) patterns in the blood headspace. An artificial intelligence nanoarray based on a varied set of chemi-sensitive nano-based structured films is developed and used to detect and stage cancer. As a proof-of-concept, three cancer models are tested showing high incidence and mortality rates in the population: breast cancer, ovarian cancer, and pancreatic cancer. The nanoarray has >84% accuracy, >81% sensitivity, and >80% specificity for early detection and >97% accuracy, 100% sensitivity, and >88% specificity for metastasis detection. Complementary mass spectrometry analysis validates these results. The ability to analyze such a complex biological fluid as blood, while considering data of many VOCs at a time using the artificially intelligent nanoarray, increases the sensitivity of predictive models and leads to a potential efficient early diagnosis and disease-monitoring tool for cancer.

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities.

Mass spectrometry-based proteomics for abiotic stress studies.

Kinase assay linked phosphoproteomics (KALIP) and universal phosphoproteomics (UP) workflows are mass spectrometry (MS)-based phosphoproteomics technologies utilized for studying abiotic stress signaling networks. KALIP consists of executing isotope-labeled phosphorylation reactions in vitro and screening in vivo phosphorylated peptides/pools of substrates. For UP, guanidine hydrochloride (GdnHCl) is used to break down the cell wall before methanol-chloroform precipitation is applied to remove interferences. Both strategies conclude with subsequent analyses of enriched phosphopeptides by complementary MS techniques to determine phosphopeptide abundance changes and protein phosphorylation sites to uncover novel signaling components in the pathway.

Arsenosugars and arsenolipids are formed simultaneously by the unicellular alga

Environmental context Arsenic is a globally distributed element, occurring in various chemical forms with toxicities ranging from harmless to highly toxic. We conducted 48-h cell culture experiments under batch and continuous conditions using the ubiquitous marine unicellular alga Dunaliella tertiolecta and evaluated the alga’s arsenic metabolome over time. We found that the alga first methylates the inorganic As taken up from the surrounding water, and then further metabolises the intermediate simultaneously into more complex organo-arsenic molecules like sugars and lipids. These time series experiments are valuable pieces in the puzzle of how algae bio-metabolise arsenic, and in our understanding of the global arsenic cycle. Rationale The uptake of arsenate by algae from oceanic waters and its transformation to arsenosugars and arsenolipids is well established, but the biosynthetic pathways remain largely unknown. Methodology We investigated these pathways by using time-series experiments over 48 h to follow the formation of organoarsenic species from arsenate-enriched medium (15 µg As L−1) by the unicellular alga Dunaliella tertiolecta cultured under batch and continuous culture conditions. We used complementary mass spectrometry methods for the determination and quantification of 14 arsenic species; an additional three species could be quantified but remained unidentified. Results The alga rapidly methylated the arsenate to dimethylarsinate (DMA), which then served as the precursor to arsenosugars and arsenolipids; the concentrations of these complex organoarsenicals increased throughout the experiments accompanied by a concomitant reduction in DMA concentrations. The pattern of compounds formed by the alga was similar for both batch and continuous cultures, but the concentrations were 2–3-fold higher in the continuous culture samples and the increases with time were much clearer. Discussion The data suggest that the arsenosugars and the arsenolipids were mostly formed simultaneously from DMA, although there was an indication that the arsenic phospholipids were at least partly also being formed from the arsenosugars. Overall, the data are consistent with a direct biosynthesis of DMA from arsenate by D. tertioleta, and thereafter a non-specific incorporation of DMA into commonly available alga metabolites encompassing various sugars and lipids.

Comparison of Chilean honeys through MALDI-TOF-MS profiling and evaluation of their antioxidant and antibacterial potential

Honey is the most famous natural sweet substance produced by honeybees (Apis mellifera). It contains numerous functional compounds, from which health benefits are obtained. In Chile, the production of honey is associated with its unique biodiversity, but it is exported mostly, as bulk honey. This work aimed to characterize the chemical and biological properties of Chilean honey on a large scale. The use of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) technique, combined with multivariate statistical analysis was introduced to study the chemical profiles of polyfloral honey. The use of a complementary mass spectrometry method allowed the identification of 25 different constituents in the studied honey, including hydrocarbons, acids, esters, glycoside isoprenoids, ketones, and a dihydroxyflavanone. The evaluation of biological properties in Chilean honey was measured in a representative number of polyfloral samples. For this purpose, the total phenolics and flavonoid content were measured in the selected samples. The antioxidant activity of the honey was evaluated through the ferric reducing antioxidant power assay (FRAP) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. On the other hand, the antibacterial activity was assessed by the dilution technique to determine the minimum inhibitory concentration (MIC) of the honey against seven bacterial strains, including Gram-positive and Gram-negative bacteria. The results demonstrated that the antioxidant and antibacterial activities of Chilean honey are variable and they could have similar properties in comparison with other well-recognized bioactive honey.

Yeast-based reference materials for quantitative metabolomics

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC–MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.

Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity.

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

Abstract 2437: Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods

Abstract The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.

Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.

Detection of Single Cancer Cells in Blood with Artificially Intelligent Nanoarray.

Detection and monitoring of single cancer cells (SCCs), such as circulating tumor cells (CTCs), would be of aid in an efficient early detection of cancer, a tailored (personalized) therapy, and in a fast bedside assessment of treatment efficacy. Nevertheless, currently available techniques, which mostly rely on the isolation of SCCs based on their physical or biological properties, suffer from low sensitivity, complicated technical procedures, low cost-effectiveness, and being unsuitable for continuous monitoring. We report here on the design and use of an artificially intelligent nanoarray based on a heterogeneous set of chemisensitive nanostructured films for the detection of SCCs using volatile organic compounds emanating in the air trapped above blood samples containing SCCs. For demonstration purposes, we have focused on samples containing A549 lung cancer cells (hereafter, SCCA549). The nanoarray developed to detect SCCA549 has >90% accuracy, >85% sensitivity, and >95% specificity. Detection works irrespective of the medium and/or the environment. These results were validated by complementary mass spectrometry. The ability to continuously record, store, and preprocess the signals increases the chances that this nanotechnology might also be useful in the early detection of cancer cells in the blood and continuous monitoring of their possible progression.

Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid‐binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo‐electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N‐linked carbohydrate post‐translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three‐dimensional architecture not seen among other retinoid‐binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.