Nature Protocols最新文献

Glycosaminoglycans (GAGs) are linear, unbranched heteropolysaccharides whose structural complexity determines their function. Accurate quantification of GAGs in biofluids at high throughput is relevant for numerous biomedical applications. However, because of the structural variability of GAGs in biofluids, existing protocols require complex pre-analytical procedures, have limited throughput and lack accuracy. Here, we describe the extraction and quantification of GAGs by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-MS/MS). Designed for 96-well plates, this method enables the processing of up to 82 study samples per plate, with the remaining 14 wells used for calibrators and controls. Key steps include the enzymatic depolymerization of GAGs, their derivatization with 2-aminoacridone and their quantification via UHPLC-MS/MS. Each plate can be analyzed in a single UHPLC-MS/MS run, offering the quantitative and scalable analysis of 17 disaccharides from chondroitin sulfate, heparan sulfate and hyaluronic acid, with a level of precision and reproducibility sufficient for their use as biomarkers. The procedure from sample thawing to initiating the UHPLC-MS/MS run can be completed in ~1.5 d plus 15 min of MS runtime per sample, and it is structured to fit within ordinary working shifts, thus making it a valuable tool for clinical laboratories seeking high-throughput analysis of GAGs. The protocol requires expertise in UHPLC-MS/MS.

Superstructures with architectural complexity and unique functionalities are promising for a variety of practical applications in many fields, including mechanics, sensing, photonics, catalysis, drug delivery and energy storage/conversion. In the past five years, a number of attempts have been made to build superparticles based on amphiphilic polymeric micelle units, but most have failed owing to their inherent poor stability. Determining how to stabilize micelles and control their superassembly is critical to obtaining the desired mesoporous superparticles. Here we provide a detailed procedure for the preparation of ultrastable polymeric monomicelle building units, the creation of a library of ultrasmall organic-inorganic nanohybrids, the modular superassembly of monomicelles into hierarchical superstructures and creation of novel multilevel mesoporous superstructures. The protocol enables precise control of the number of monomicelle units and the derived mesopores for superparticles. We show that ultrafine nanohybrids display enhanced mechanical antipressure performance compared with pristine polymeric micelles, and describe the functional characterization of mesoporous superstructures that exhibit excellent oxygen reduction reactivity. Except for the time (4.5 d) needed for the preparation of the triblock polystyrene-block-poly(4-vinylpyridine)-block-poly(ethylene oxide) PS-PVP-PEO or the polystyrene-block-poly(acrylic acid)-block-poly(ethylene oxide) (PS-PAA-PEO) copolymer, the synthesis of the ultrastable monomicelle, ultrafine organic-inorganic nanohybrids, hierarchical superstructures and mesoporous superparticles require ~6, 30, 8 and 24 h, respectively. The time needed for all characterizations and applications are 18 and 10 h, respectively.

Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.