High-throughput proteomics based on mass spectrometry (MS) analysis has permeated biomedical science and propelled numerous research projects. pFind 3 is a database search engine for high-speed and in-depth proteomics data analysis. pFind 3 features a swift open search workflow that is adept at uncovering less obvious information such as unexpected modifications or mutations that would have gone unnoticed using a conventional data analysis pipeline. In this protocol, we provide step-by-step instructions to help users mastering various types of data analysis using pFind 3 in conjunction with pParse for data pre-processing and if needed, pQuant for quantitation. This streamlined pParse-pFind-pQuant workflow offers exceptional sensitivity, precision, and speed. It can be easily implemented in any laboratory in need of identifying peptides, proteins, or post-translational modifications, or of quantitation based on ¹⁵N-labeling, SILAC-labeling, or TMT/iTRAQ labeling.

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

With the biological relevance of the whole cells, low cost compared with animal experiments, a wide variety of cell-based screening platforms (cell-based assay, cell-based microfluidics, cell-based biosensor, cell-based chromatography) have been developed to address the challenges of drug discovery. In this review, we conclude the current advances in cell-based screening and summary the pros and cons of the platforms for different applications. Challenges and improvement strategies associated with cell-based methods are also discussed.

Cells and tissues are exquisitely organized in a complex but ordered manner to form organs and bodies so that individuals can function properly. The spatial organization and tissue architecture represent a keynote property underneath all living organisms. Molecular architecture and cellular composition within intact tissues play a vital role in a variety of biological processes, such as forming the complicated tissue functionality, precise regulation of cell transition in all living activities, consolidation of central nervous system, cellular responses to immunological and pathological cues. To explore these biological events at a large scale and fine resolution, a genome-wide understanding of spatial cellular changes is essential. However, previous bulk RNA sequencing and single-cell RNA sequencing technologies could not obtain the important spatial information of tissues and cells, despite their ability to detect high content transcriptional changes. These limitations have prompted the development of numerous spatially resolved technologies which provide a new dimension to interrogate the regional gene expression, cellular microenvironment, anatomical heterogeneity and cell-cell interactions. Since the advent of spatial transcriptomics, related works that use these technologies have increased rapidly, and new methods with higher throughput and resolution have grown quickly, all of which hold great promise to accelerate new discoveries in understanding the biological complexity. In this review, we briefly discussed the historical evolution of spatially resolved transcriptome. We broadly surveyed the representative methods. Furthermore, we summarized the general computational analysis pipeline for the spatial gene expression data. Finally, we proposed perspectives for technological development of spatial multi-omics.

Nanomaterials-based artificial enzymes (nanozymes) with valuable enzyme-like catalytic properties have been booming during the past few years. Promoted by the advances in biological medicine and nanotechnology, nanozymes possess the potential to serve as an emerging agent for biosensing, immunoassays, detection and diagnosis, catalytic therapeutics, and other applications in the biomedicine field. Two-dimensional (2D) nanomaterials are of considerable interest in biomedical applications due to their ultrathin layered structure and unique physiochemical properties. Inspired by the diversified catalytic performance of 2D nanomaterials, scientists extensively have developed 2D materials as bioactive nanozymes for theranostic nanomedicine. Here, recent advances in enzyme-like 2D nanomaterials design and construction are comprehensively presented. Additionally, we exhibit that, with the synergistic effect of catalytic activities and desirable physicochemical performances, 2D nanozymes can serve as versatile platforms with extensive applications from target detection to in vivo theranostic. It is believed that such promising alternatives towards natural enzymes will be of vital significance in the field of nanotechnology and biomedicine.

Liquid–liquid phase separation (LLPS) is an emerging phenomenon involved in various biological processes. The formation of phase-separated condensates is crucial for many intrinsically disordered proteins to fulfill their biological functions. Using the recombinant protein to reconstitute the formation of condensates in vitro has become the standard method to investigate the behavior and function of LLPS. Meanwhile, there is an urgent need to characterize the LLPS in living cells. Importantly, condensates formed through LLPS at physical relevant concentrations are often smaller than the optical diffraction limit, which makes precise characterization and quantification inaccurate due to the scatter of light. The booming development of super-resolution optical microscopy enables the visualization of multiple obscured subcellular components and processes, which is also suitable for the LLPS research. In this protocol, we provide step-by-step instructions to help users take advantage of super-resolution imaging to depict the morphology and quantify the molecule number of endogenous condensates in living cells using RNA Pol II as an example. This streamlined workflow offers exceptional robustness, sensitivity, and precision, which could be easily implemented in any laboratory with an inverted total internal reflection microscope. We expect that super-resolution microscopy will contribute to the investigation of both large and tiny condensates under physiological and pathological conditions and lead our understanding of the mechanism of LLPS to a higher and deeper layer.

Single-molecule methods have been applied to study the mechanisms of many biophysical systems that occur on the nanometer scale. To probe the dynamics of such systems including vesicle docking, tethering, fusion, trafficking, protein-membrane interactions, etc., and to obtain reproducible experimental data; proper methodology and framework are crucial. Here, we address this need by developing a protocol for immobilization of vesicles composed of synthetic lipids and measurement using total internal reflection fluorescence (TIRF) microscopy. Furthermore, we demonstrate applications including vesicle clustering mediated by proteins such as alpha-Synuclein (αSyn) and the influence of external ions by using TIRF microscopy. Moreover, we use this method to quantify the dependence of lipid composition and charge on vesicle clustering mediated by αSyn which is based on the methods previously reported.

Intracellular transport is the basis for the transfer of matter, energy, and information in cells and is critical to many cellular functions. Within the nonequilibrium environment of living cells, the transport behaviours are far from the traditional motion in liquid but are more complex and active. With the advantage of high spatial and temporal resolution, the single-particle tracking (SPT) method is widely utilized and has achieved great advances in revealing intracellular transport dynamics. This review describes intracellular transport from a physical perspective and classifies it into two modes: diffusive motion and directed motion. The biological functions and physical mechanisms for these two transport modes are introduced. Next, we review the principle of SPT and its advances in two aspects of intracellular transport. Finally, we discuss the prospect of near infrared SPT in exploring the in vivo intracellular transport dynamics.

Cell membranes are complicated multicomponent structures, related to many basic cellular processes, such as substance transporting, energy conversion, signal transduction, mechanosensing, cell adhesion and so on. However, cell membranes have long been difficult to study at a single-molecule level due to their complex and dynamic properties. During the last decades, biophysical imaging techniques, such as atomic force microscopy and super-resolution fluorescent microscopy, have been developed to study biological structures with unprecedented resolution, enabling researchers to analyze the composition and distribution of membrane proteins and monitor their specific functions at single cell/molecule level. In this review, we highlight the structure and functions of cell membranes based on up-to-date biophysical techniques. Additionally, we describe the recent advances in force-based detecting technology, which allow insight into dynamic events and quantitativelymonitoring kinetic parameters for trans-membrane transporting in living cells.

Force spectroscopy experiments use mechanical force as a control factor to regulate the folding and unfolding process of proteins. Atomic force microscopy has been widely used to study the mechanical stability of proteins, and obtained unfolding forces and unfolding distance of different proteins, while recently, more low force folding and unfolding measurements were done by optical tweezers and magnetic tweezers. Due to the relatively small distortion of the free energy landscape, low force measurements give the free energy landscape information over bigger conformational space. In this review, we summarize the results of force spectroscopy experiments on different proteins. The unfolding distance obtained at high forces by atomic force microscopy are mostly smaller than 2 nm, while the unfolding distances at low forces distribute over a larger range: from a negative value to more than 6 nm. The sizes of the transition states at low force are ~4 nm for most compact two-state globular proteins, which indicates that this transition state might be the general free energy barrier separating the unfolded state and the theoretically predicated molten globule state. Up to now, only a limited number of proteins has been studied at low forces. We expect that more and more proteins with different conformations will be studied at low forces to reveal the general protein folding mechanism.

Fluorescence microscopy has become a routine tool in biology for interrogating life activities with minimal perturbation. While the resolution of fluorescence microscopy is in theory governed only by the diffraction of light, the resolution obtainable in practice is also constrained by the presence of optical aberrations. The past two decades have witnessed the advent of super-resolution microscopy that overcomes the diffraction barrier, enabling numerous biological investigations at the nanoscale. Adaptive optics, a technique borrowed from astronomical imaging, has been applied to correct for optical aberrations in essentially every microscopy modality, especially in super-resolution microscopy in the last decade, to restore optimal image quality and resolution. In this review, we briefly introduce the fundamental concepts of adaptive optics and the operating principles of the major super-resolution imaging techniques. We highlight some recent implementations and advances in adaptive optics for active and dynamic aberration correction in super-resolution microscopy.

Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.

Protein amyloid fibrillation, a process of liquid to solid phase transition, is involved in the pathogenesis of a variety of human diseases. Several amyloid proteins including α-synuclein (α-syn), Tau, amyloid β (Aβ) protein, and TAR DNA-binding protein 43 kDa (TDP-43) form pathological fibrils and deposit in patient brains of different neurodegenerative diseases (NDs) such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and Amyotrophic lateral sclerosis (ALS). Preparation and characterization of amyloid fibrils in vitro are essential for studying the molecular mechanism underlying the dynamic amyloid aggregation and its pathogenesis in diseases. In this protocol, we take PD-associated α-syn as an example, and describe amyloid protein purification and fibrillation approaches. We then introduce biochemical and biophysical characterization of amyloid fibrils by Thioflavin-T (ThT) fluorescence kinetics assay, transmission electron microscopy (TEM), atomic force microscopy (AFM) and multiple fibril stability measurement assays. The approaches described here are applicable to different amyloid proteins, and are of importance for further study on the structure determination of amyloid fibrils and their pathological function in cells and animal models.

Complex physical cues including two-dimensional membrane environment, dynamic mechanical force, and bioelectric activity inevitably affect membrane receptor functions. Multiplexed single-molecule force spectroscopy (SMFS) techniques with the capability of live-cell measurements are essential to systemically dissect receptor’s functions under complex biophysical regulation. In this review, we summarize recent progress of live-cell based SMFS techniques and specifically focus on the progress of SMFS on the biomembrane force probe with enhanced mechanical stability and multiplexed capability of fluorescence imaging. We further suggest the necessity of developing multiplexed SMFS techniques with simultaneous bioelectric regulation capability to investigate membrane potential regulated membrane receptor functions. These state-of-art multiplexed SMFS techniques will dissect membrane receptors functions in a systematic biophysical angle, resolving the biochemical, biomechanical and bioelectrical regulatory mechanisms in physiologically relevant conditions.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool to explore cells. With an increasing number of scRNA-seq data analysis tools that have been developed, it is challenging for users to choose and compare their performance. Here, we present an overview of the workflow for computational analysis of scRNA-seq data. We detail the steps of a typical scRNA-seq analysis, including experimental design, pre-processing and quality control, feature selection, dimensionality reduction, cell clustering and annotation, and downstream analysis including batch correction, trajectory inference and cell–cell communication. We provide guidelines according to our best practice. This review will be helpful for the experimentalists interested in analyzing their data, and will aid the users seeking to update their analysis pipelines.

Liquid–liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.