Nature Protocols最新文献

The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography-high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH^- ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS.

Ordered mesoporous metal oxides (OMMOs) with periodically interconnected mesopores and crystalline framework have attracted ever-growing attention due to their high specific surface area, well-defined mesoscopic structures and adjustable pore-wall chemical microenvironment. It has been difficult to rationally design OMMO syntheses because the hydrolysis of metal salts is difficult to control; it is also difficult to find precursors that have a strong enough interaction with the structure-directing agents and oxides to overcome the formation of disordered metal oxide crystals rather than frameworks at the temperatures required for calcination. Here we describe an evaporation-induced cooperative assembly (EICA) approach for the controllable synthesis of high-quality OMMOs (for example, WO₃). The EICA approach endows precise control over the intermolecular interactions between metal oxide precursors and amphiphilic block copolymers such as poly(ethylene oxide)-block-polystyrene through ligand-assisted or cluster-involved assembly strategies and optimizes the thermal treatment process through carbon-supported crystallization. Based on this Protocol, a library of OMMOs with different framework compositions and desired pore sizes (10-35 nm, by changing the polystyrene length of poly(ethylene oxide)-block-polystyrene template) can be readily tuned, which can be further precisely modified by pore-wall engineering (for example, element doping, noble metal decoration and heterojunction construction). We describe the detailed experimental design and synthesis procedures to ensure the reproducibility of the experiments. Chemiresistive gas sensing and electrocatalytic hydrogen evolution reaction are introduced as potential applications of OMMOs. Except for the time (~2.5 d) needed for the preparation of amphiphilic block copolymers, the EICA approach for synthesizing OMMOs requires ~3.5 d without requiring special expertise.

The rapid acceleration of global electrification has increased demand for sustainable energy storage, making lithium-ion batteries (LIBs) essential for various applications. However, their limited lifespan presents challenges related to resource waste and environmental risks. Unlike traditional metallurgical methods, which extract key metals from spent cathodes, the direct recycling process repairs damaged materials, maximizing their residual value through effective treatments. Despite widespread interest, systematic protocols to guide interdisciplinary researchers in direct recycling studies remain scarce. Using spent LiMn₂O₄ as an example, this protocol outlines a general approach for direct recycling and upcycling of spent LIBs. Initially, the failure condition of the spent cathode is evaluated using X-ray diffraction and inductively coupled plasma analysis to determine appropriate recycling parameters. The resulting recycled products include regenerated LiMn₂O₄ and upcycled next-generation cathode materials, such as high-voltage LiNi_0.5Mn_1.5O₄ and Co-free, Li-rich Li_1.2Ni_0.2Mn_0.6O₂. Subsequently, electron microscopy, spectroscopic techniques and electrochemical performance tests evaluate recycling effectiveness. This protocol incorporates two representative recycling methods to provide readers with a detailed procedural guide. Solid-phase regeneration forms the basis of most direct recycling technologies; thus, it requires minimal adjustments for broad applicability. Joule heating, a more emerging recycling technology, leverages rapid nonequilibrium reactions, substantially reducing processing time and introducing beneficial structural defects and elemental gradient distributions within the material. Compared to metallurgical methods, solid-phase and Joule heating-based protocols reduce recycling time to ~32 h and 5 h, respectively. Overall, this protocol provides a reliable guide for researchers, promoting sustainable LIB recycling and advancing clean energy research.

Prime editing is a versatile genome editing technology that enables precise genetic modifications without inducing DNA double-strand breaks. Owing to numerous variables in the prime editing guide RNA (pegRNA) design, experimentally identifying the most efficient pegRNA for a specific locus and edit is laborious. Therefore, we have developed computational tools to streamline this process. Here we present a comprehensive protocol detailing how to use PRIDICT2.0 and ePRIDICT, machine-learning models that assess the influence of the pegRNA design and chromatin context on prime editing. PRIDICT2.0 is an ensemble of attention-based bidirectional recurrent neural networks that predicts pegRNA efficiencies for replacements, insertions or deletions in different cellular contexts. Compared with other pegRNA design tools, PRIDICT2.0 accommodates larger edits-up to 40 base pairs-across diverse edit types, also allowing the introduction of silent bystander edits that can enhance editing efficiency. ePRIDICT, a gradient-boosting algorithm, further accounts for the local chromatin environments and assesses how the genomic location of the target site affects prime editing rates. Both tools are available at www.pridict.it for individual predictions or can be installed locally for batch processing of multiple edits and target sites. The protocol provides step-by-step instructions on using PRIDICT2.0 and ePRIDICT, covering sequence input, prediction generation and interpretation. Web-based predictions take under a minute, while local installation and batch processing may take up to several hours, depending on the dataset size. By streamlining pegRNA selection and chromatin context analysis, these tools promote the adoption of prime editing in basic and translational research.

Human primary fetal stem cell-derived organoids are used to model developing tissues in vitro. However, ethical and legislative constraints restrict fresh fetal tissue collection in several countries. Amniotic fluid (AF) is easily accessible with minimal ethical and regulatory constraints for collection. Our team recently showed that tissue-specific stem/progenitor cells can be isolated from fetal fluids collected during pregnancy through clinically indicated minimally invasive procedures conducted during the second and third trimesters. These samples consistently generate fetal lung, kidney tubule and gastrointestinal epithelial organoids autologous to the developing fetus. AF-derived organoids (AFOs) allow the investigation of fetal epithelia at developmentally relevant stages. Moreover, AFOs allow research to be conducted on late gestational stages, hardly accessible with other methods. Here, we provide a detailed protocol to establish, characterize and cryopreserve AFOs from viable AF cells. This includes the processing of patient-derived AF samples, viable cell sorting, seeding, establishment of clonal AFO lines, tissue phenotyping, expansion and cryopreservation. Additionally, we describe a straightforward immunofluorescence-based approach to pinpoint the tissue identity of the AFOs in a quick and cost-effective manner. In our hands, the protocol enabled the generation of primary fetal AFOs from 85.71% of samples (62.5% ascribed to the fetal lung, 59.4% to the kidney tubule and 6.2% to the small intestine). It takes 4-6 weeks to implement, requiring only standard equipment and expertise commonly available in cell biology laboratories.

Methanogenic archaea play a crucial role in the global carbon cycle and in climate change. Recent metagenomic sequencing has revealed a considerable number of (putative) H₂-dependent methylotrophic methanogens (HMMs) across the archaeal tree and in diverse environments. Traditional isolation methods, such as dilution-to-extinction and roll-tube techniques, fail to cultivate fastidious HMMs. Here, we describe a four-stage isolation strategy designed to selectively isolate HMMs by using a flexible combination of methods to systematically reduce microbial complexity to a pure culture. In the initial stage, the growth conditions for the target HMM were optimized through closed-batch cultivation encompassing >50 conditions. Second, HMM-containing cultures were serially diluted in 96-well plates combined with substrate limitation to eliminate non-target archaea. In stage 3, the bacterial diversity in the culture was further decreased to a single bacterium by treatment with antibiotics and lysozyme. Finally, a last bacterial contaminant was removed by repeated addition of antibiotic mixtures and successive dilution transfers, leading to the successful isolation of the first pure culture of Methanosuratincola petrocarbonis LWZ-6, an HMM of the phylum Thermoproteota. This protocol also describes molecular methods, including 16S rRNA gene amplicon sequencing, metagenome sequencing and quantitative PCR, to track microbial community shifts and assess the growth advantage of the target HMM, enabling monitoring of the stepwise elimination of non-target microorganisms and ultimately confirming the purification of the target HMM. The duration of the protocol will vary for different HMMs depending on their substrate utilization, growth rate and method selection.

Hydrogen peroxide (H₂O₂) is a natural product of aerobic metabolism. It acts as a signaling molecule and regulates fundamental cellular functions. However, it has remained difficult to measure intracellular H₂O₂ with high specificity and in a quantitative manner. Here, we present a detailed protocol for a chemogenetic method that enables the detection and quantitation of H₂O₂ in living cells by converting intracellular H₂O₂ into fluorescent or luminescent signals. This is achieved by expressing the engineered heme peroxidase APEX2 in cells and subcellular locations of interest and by providing an appropriate fluorogenic or luminogenic substrate from outside. This method differs fundamentally from previously developed genetically encoded H₂O₂ probes; those are reversible and measure the balance between probe thiol oxidation and reduction. By contrast, APEX2 turns over its substrate irreversibly and therefore directly measures endogenous H₂O₂ availability. Our detailed step-by-step protocol covers the generation of APEX2-expressing cell lines, the implementation of fluorescent and luminescent measurements and examples for application. Ectopic expression of APEX2 can be achieved in 3 days, while the actual measurements typically require 1-2 h. This protocol is intended for entry-level scientists.