Nature Protocols最新文献

{"title":"Quantifying genome-wide transcription factor binding affinities for chromatin using BANC-seq","authors":"Roelof A. Wester, Hannah K. Neikes, Rik G. H. Lindeboom, Michiel Vermeulen","doi":"10.1038/s41596-024-01026-7","DOIUrl":"10.1038/s41596-024-01026-7","url":null,"abstract":"Transcription factors (TFs) bind specific DNA sequences to regulate transcription. Apart from DNA sequences, local factors such as DNA accessibility and chromatin structure determine the affinity of a TF for any given locus. Including these factors when measuring TF–DNA affinities has proven difficult. To address this challenge, we recently developed a method called binding affinities in native chromatin by sequencing (BANC-seq). In BANC-seq, intact mammalian nuclei are incubated with a concentration range of epitope-tagged TF, followed by either chromatin immunoprecipitation or cleavage under target and release using nuclease with spike-in DNA. This allows determination of apparent dissociation constant (KdApp) values, defined by the concentration of TF at which half-maximum binding occurs, across the genome. Here we present a detailed stepwise protocol for BANC-seq, including downstream data analysis. In principle, any molecular biologist should be able to perform a BANC-seq experiment in as little as 1.5 d (excluding analysis). However, preprocessing and analysis of the sequencing data does require some experience in command-line shell and R programming. BANC-seq enables quantification of genome-wide transcription factor binding affinities in the native chromatin context. This protocol describes implementations based on chromatin immunoprecipitation or cleavage under target and release using nuclease, followed by library preparation, sequencing and data analysis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3590-3612"},"PeriodicalIF":13.1,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141855984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Host circuit engagement of human cortical organoids transplanted in rodents","authors":"Kevin W. Kelley, Omer Revah, Felicity Gore, Konstantin Kaganovsky, Xiaoyu Chen, Karl Deisseroth, Sergiu P. Pașca","doi":"10.1038/s41596-024-01029-4","DOIUrl":"10.1038/s41596-024-01029-4","url":null,"abstract":"Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell–derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30–60 days, and the transplantation procedure itself requires ~0.5–1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron–host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages. The transplantation of human cortical organoids in rats enables maturation and integration of human neural cells that can engage with the host circuitry, providing a framework to study alterations in morphology and physiology of patient-derived tissue.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3542-3567"},"PeriodicalIF":13.1,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-resolution topographic surveying and change detection with the iPhone LiDAR","authors":"Gregor Luetzenburg, Aart Kroon, Kristian K. Kjeldsen, Kristen D. Splinter, Anders A. Bjørk","doi":"10.1038/s41596-024-01024-9","DOIUrl":"10.1038/s41596-024-01024-9","url":null,"abstract":"This paper introduces a comprehensive protocol leveraging open-access techniques to create small- to medium-scale 3D representations of the environment by using iPhone and iPad light detection and ranging (LiDAR). The protocol focuses on two capabilities of the iPhone LiDAR. The first capability is 3D modeling: iPhone LiDAR rapidly generates detailed indoor and outdoor 3D models, providing insights into object size, volume and geometry. The second capability is change detection: the 3D models created by the LiDAR sensor can be used for precise measurement of changes over time. Compared to other 3D topographic surveying methods, this method is rapid, high resolution, low cost and easy to use. The protocol outlines iPhone LiDAR scanning practices, model export and change detection. The expected results after executing the protocol are (i) a detailed 3D model of a small- to medium-sized object or area of interest and (ii) a distance point cloud revealing change between two point clouds of the same object or area between different times. The entire protocol can be conducted within 2 h by anyone with an iPhone with the LiDAR sensor and a computer. This protocol empowers scientists, students and community members conducting research with a cheap, easy-to-use method for addressing a range of questions and challenges, thus benefiting experts and the broader community. This protocol describes the use of iPhone LiDAR to generate high-resolution 3D models for topographic surveying and to measure their changes over time. The protocol simplifies the use of the iPhone LiDAR sensor, making it accessible to non-experts.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3520-3541"},"PeriodicalIF":13.1,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A network control theory pipeline for studying the dynamics of the structural connectome","authors":"Linden Parkes, Jason Z. Kim, Jennifer Stiso, Julia K. Brynildsen, Matthew Cieslak, Sydney Covitz, Raquel E. Gur, Ruben C. Gur, Fabio Pasqualetti, Russell T. Shinohara, Dale Zhou, Theodore D. Satterthwaite, Dani S. Bassett","doi":"10.1038/s41596-024-01023-w","DOIUrl":"10.1038/s41596-024-01023-w","url":null,"abstract":"Network control theory (NCT) is a simple and powerful tool for studying how network topology informs and constrains the dynamics of a system. Compared to other structure–function coupling approaches, the strength of NCT lies in its capacity to predict the patterns of external control signals that may alter the dynamics of a system in a desired way. An interesting development for NCT in the neuroscience field is its application to study behavior and mental health symptoms. To date, NCT has been validated to study different aspects of the human structural connectome. NCT outputs can be monitored throughout developmental stages to study the effects of connectome topology on neural dynamics and, separately, to test the coherence of empirical datasets with brain function and stimulation. Here, we provide a comprehensive pipeline for applying NCT to structural connectomes by following two procedures. The main procedure focuses on computing the control energy associated with the transitions between specific neural activity states. The second procedure focuses on computing average controllability, which indexes nodes’ general capacity to control the dynamics of the system. We provide recommendations for comparing NCT outputs against null network models, and we further support this approach with a Python-based software package called ‘network control theory for python’. The procedures in this protocol are appropriate for users with a background in network neuroscience and experience in dynamical systems theory. This protocol describes a comprehensive framework for applying network control theory to the human structural connectome to study its topology and show how that topology affects the dynamics of neural activity states, using a software package written in Python.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3721-3749"},"PeriodicalIF":13.1,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro culture of cynomolgus monkey embryos from blastocyst to early organogenesis","authors":"Yanhong Xu, Jinglei Zhai, Hao Wu, Hongmei Wang","doi":"10.1038/s41596-024-01025-8","DOIUrl":"10.1038/s41596-024-01025-8","url":null,"abstract":"Human early embryonic development is the cornerstone of a healthy baby. Abnormal early embryonic development may lead to developmental and pregnancy-related disorders. Accordingly, understanding the developmental events and mechanisms of human early embryonic development is very important. However, attempts to reveal these events and mechanisms are greatly hindered by the extreme inaccessibility of in vivo early human embryos. Fortunately, the emergence of in vitro culture (IVC) systems for mammalian embryos provides an alternative strategy. In recent years, different two-dimensional and three-dimensional IVC systems have been developed for human embryos. Ethical limitations restrict the IVC of human embryos beyond 14 days, which makes non-human primate embryos an ideal model for studying primate developmental events. Different culture systems have supported the development of monkey embryos to days postfertilization 14 and 25, respectively. The successful recapitulation of in vivo developmental events by these IVC embryos has greatly enriched our understanding of human early embryonic development, which undoubtedly helps us to develop possible strategies to predict or treat various gestation-related diseases and birth defects. In this protocol, we establish different two-dimensional and three-dimensional IVC systems for primate embryos, provide step-by-step culture procedures and notes, and summarize the advantages and limitations of different culture systems. Replicating this protocol requires a moderate level of experience in mammalian embryo IVC, and the embryo culture requires strict adherence to the procedures we have described. This protocol describes the in vitro culture of cynomolgus monkey embryos from blastocyst to early organogenesis. Given the ethical limitations restricting the use of human embryos beyond 14 days, nonhuman primate embryos are an ideal model.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3677-3696"},"PeriodicalIF":13.1,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141766742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of nanomaterial biocoronas in biological and environmental surroundings","authors":"Peng Zhang, Mingjing Cao, Andrew J. Chetwynd, Klaus Faserl, Fazel Abdolahpur Monikh, Wei Zhang, Rawi Ramautar, Laura-Jayne A. Ellis, Hossein Hayat Davoudi, Katie Reilly, Rong Cai, Korin E. Wheeler, Diego Stéfani Teodoro Martinez, Zhiling Guo, Chunying Chen, Iseult Lynch","doi":"10.1038/s41596-024-01009-8","DOIUrl":"10.1038/s41596-024-01009-8","url":null,"abstract":"A biomolecular coating, or biocorona, forms on the surface of engineered nanomaterials (ENMs) immediately as they enter biological or environmental systems, defining their biological and environmental identity and influencing their fate and performance. This biomolecular layer includes proteins (the protein corona) and other biomolecules, such as nucleic acids and metabolites. To ensure a meaningful and reproducible analysis of the ENMs-associated biocorona, it is essential to streamline procedures for its preparation, separation, identification and characterization, so that studies in different labs can be easily compared, and the information collected can be used to predict the composition, dynamics and properties of biocoronas acquired by other ENMs. Most studies focus on the protein corona as proteins are easier to monitor and characterize than other biomolecules and play crucial roles in receptor engagement and signaling; however, metabolites play equally critical roles in signaling. Here we describe how to reproducibly prepare and characterize biomolecule-coated ENMs, noting especially the steps that need optimization for different types of ENMs. The structure and composition of the biocoronas are characterized using general methods (transmission electron microscopy, dynamic light scattering, capillary electrophoresis–mass spectrometry and liquid chromatography–mass spectrometry) as well as advanced techniques, such as transmission electron cryomicroscopy, synchrotron-based X-ray absorption near edge structure and circular dichroism. We also discuss how to use molecular dynamic simulation to study and predict the interaction between ENMs and biomolecules and the resulting biocorona composition. The application of this protocol can provide mechanistic insights into the formation, composition and evolution of the ENM biocorona, ultimately facilitating the biomedical and agricultural application of ENMs and a better understanding of their impact in the environment. Engineered and anthropogenic nanoscale materials in the environment acquire a coating of biomolecules (biocorona) that modulates their properties, uptake and biodistribution. This protocol streamlines biocorona analysis to support the development of safe and sustainable nanotechnology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 10","pages":"3000-3047"},"PeriodicalIF":13.1,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141752146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Surface nanocoating of bacteria as a versatile platform to develop living therapeutics","authors":"Zhenping Cao, Jinyao Liu","doi":"10.1038/s41596-024-01019-6","DOIUrl":"10.1038/s41596-024-01019-6","url":null,"abstract":"Bacteria have been extensively utilized as living therapeutics for disease treatment due to their unique characteristics, such as genetic manipulability, rapid proliferation and specificity to target disease sites. Various in vivo insults can, however, decrease the vitality of dosed bacteria, leading to low overall bioavailability. Additionally, the innate antigens on the bacterial surface and the released toxins and metabolites may cause undesired safety issues. These limitations inevitably result in inadequate treatment outcomes, thereby hindering the clinical transformation of living bacterial therapeutics. Recently, we have developed a versatile platform to prepare advanced living bacterial therapeutics by nanocoating bacteria individually via either chemical decoration or physical encapsulation, which can improve bioavailability and reduce side effects for enhanced microbial therapy. Here we use interfacial self-assembly to prepare lipid membrane-coated bacteria (LCB), exhibiting increased resistance against a variety of harsh environmental conditions owing to the nanocoating’s protective capability. Meanwhile, we apply mechanical extrusion to generate cell membrane-coated bacteria (CMCB), displaying improved biocompatibility owing to the nanocoating’s shielding effect. We describe their detailed preparation procedures and demonstrate the expected functions of the coated bacteria. We also show that following oral delivery and intravenous injection in mouse models, LCB and CMCB present appealing potential for treating colitis and tumors, respectively. Compared with bioengineering that lacks versatile molecular tools for heterogeneous expression, the surface nanocoating technique is convenient to introduce functional components without restriction on bacterial strain types. Excluding bacterial culture, the fabrication of LCB takes ~2 h, while the preparation of CMCB takes ~5 h. This protocol adds a surface nanocoating to bacteria to improve bioavailability and reduce side effects for enhanced microbial therapy. Living bacterial therapeutics are produced by nanocoating bacteria individually via either chemical decoration or physical encapsulation.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3162-3190"},"PeriodicalIF":13.1,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141752147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics","authors":"Seung Won Shin, Prakriti Mudvari, Shravan Thaploo, Michael A. Wheeler, Daniel C. Douek, Francisco J. Quintana, Eli A. Boritz, Adam R. Abate, Iain C. Clark","doi":"10.1038/s41596-024-01021-y","DOIUrl":"10.1038/s41596-024-01021-y","url":null,"abstract":"Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called ‘focused interrogation of cells by nucleic acid detection and sequencing’ (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence. FIND-seq is a nucleic acid cytometry platform capable of isolating rare cells on the basis of RNA or DNA markers. This protocol outlines the molecular and microfluidic steps to perform FIND-seq, followed by bulk or single-cell transcriptomic analysis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3191-3218"},"PeriodicalIF":13.1,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141741617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test","authors":"Ting Zhang, Yuxi Wang, Xucong Teng, Ruijie Deng, Jinghong Li","doi":"10.1038/s41596-024-01022-x","DOIUrl":"10.1038/s41596-024-01022-x","url":null,"abstract":"The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30–45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants. The authors present MARVE, a paper-based, preamplification-free diagnostic tool for fast but accurate colorimetric detection of viral variants at single-nucleotide resolution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3426-3455"},"PeriodicalIF":13.1,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141724008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multi-glycomic platform for the analysis of food carbohydrates","authors":"Garret Couture, Shawn Ehlers Cheang, Christopher Suarez, Ye Chen, Nikita P. Bacalzo Jr, Jiani Jiang, Cheng-Yu Charlie Weng, Aaron Stacy, Juan J. Castillo, Omar Delannoy-Bruno, Daniel M. Webber, Michael J. Barratt, Jeffrey I. Gordon, David A. Mills, J. Bruce German, Naomi K. Fukagawa, Carlito B. Lebrilla","doi":"10.1038/s41596-024-01017-8","DOIUrl":"10.1038/s41596-024-01017-8","url":null,"abstract":"Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3321-3359"},"PeriodicalIF":13.1,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141724007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}