Buffer Composition Research Articles

Towards Accurate Biocompatibility: Rethinking Cytotoxicity Evaluation for Biodegradable Magnesium Alloys in Biomedical Applications

Magnesium and its alloys represent promising candidates for biomedical implants due to their biodegradability and mechanical properties, which are similar to natural bone. However, their rapid degradation process characterized by dynamic pH fluctuations and significant hydrogen gas evolution during biocorrosion adversely affects both in vitro and in vivo assessments. While the ISO 10993-5 and 12 standards provide guidelines for evaluating the in vitro biocompatibility of biodegradable materials, they also introduce testing variability conditions that yield inconsistent results. To address these inherent characteristics of Mg alloys, developing improved methods that accurately simulate the physiological environment for in vitro biocompatibility testing is essential. This study introduces two novel extraction approaches for evaluating Mg alloys: a buffered solution utilizing PBS/DMEM with quaternary dilutions and a modified ISO standard protocol employing decuple dilution of conventional unbuffered extracts. The present findings establish that controlled optimization of extraction conditions, specifically buffer composition and dilution parameters, enables reliable in vitro cytotoxicity assessment of Mg alloys, providing a robust methodology that advances the preclinical evaluation of these promising biodegradable materials.

A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery

Biopanning methods to select target-specific Nanobodies® (Nbs) involve presenting the antigen, immobilized on plastic plates or magnetic beads, to Nb libraries displayed on phage. Most routines are operator-dependent, labor-intensive and often material- and time-consuming. Here we validate an improved panning strategy that uses biosensors to present the antigen to phage-displayed Nbs in a well. The use of automated Octet biolayer interferometry sensors (Sartorius) enables high throughput and precise control over each step. By playing with association and dissociation times and buffer composition, one can efficiently decrease the background of aspecific and low-affinity Nbs, reducing the rounds of panning needed for the enrichment of high-affinity binders. Octet panning also enables the use of unpurified target proteins and unpurified phage from a bacterial culture supernatant. Additionally, downscaling to a 384-well format significantly reduces the amount of protein required. Moreover, enrichment of binders can be quantified by monitoring phage binding to the target by interferometry, omitting additional phage titration steps. Routinely, up to three rounds of Octet panning can be performed in only five days to deliver target-specific binders, ready for screening and characterization using the same Octet instrument.

Self‐Driving Lab for Solid‐Phase Extraction Process Optimization and Application to Nucleic Acid Purification

Sorptive bioprocesses are the basis for numerous biotechnological applications such as enzyme immobilization, biosensors, controlled drug delivery, water treatment, and molecular purification. Yet due to the complexity of these processes, their optimization is still time, labor, and cost‐intensive. This research presents a flexible self‐driving laboratory (SDL) designed for the accelerated development and optimization of solid‐phase extraction processes. As a use case, the SDL was used to optimize a DNA purification process using silica magnetic beads. Through the integration of robotics, machine learning, and data‐driven experimentation, the SDL demonstrates a highly accelerated process optimization with minimal human intervention. In the multistep purification approach, the system is able to optimize buffer compositions for DNA extraction from complex samples, demonstrating effectiveness in both conventional chaotropic salt‐based methods and innovative chaotropic salt‐free buffers. The study highlights the SDL's capability to autonomously refine process parameters, achieving significant enhancements in yield and purity of the product. This blueprint for future self‐driving optimization of bioprocess parameters showcases the potential of autonomous systems to revolutionize biochemical process development, offering insights into scalable, environmentally sustainable, and cost‐effective solutions.

CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences

The B DNA of the genome contains numerous sequences that can form various noncanonical structures including G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating-motif (i-motif, iM) formed by alternately arranged C-C+ pairs. One of the easy yet sensitive methods to study G4s and iMs is circular dichroism (CD) spectroscopy which generates characteristic G4 and iM peaks. We have analyzed and compared the effects of various environmental factors including pH, buffer composition, temperature, flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB) on the CD patterns of G4s and iMs generated by two groups of DNA molecules, one containing tandem repeats of GGGGCC and CCCCGG from the C9ORF72 gene associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and the second containing polyG/polyC clusters from oncogene promoter-proximal regions without such tandem repeats. Changes in pH caused drastic changes in CCCCGG-iM and GGGGCC-G4 and the changes were dependent on repeat numbers and G:C base-pairing. In contrast, with the DNA sequences from the promoter-proximal regions of oncogenes, iMs disassembled upon pH changes with the peak slowly shifting to lower wavelength but the G4s did not show significant change. Complementary DNA strands and flanking DNA sequences also regulate G4 and iM formation. The single-stranded DNA binding protein, SSB, disassembled both G4s and iMs formed by almost all sequences suggesting an in vivo role for SSBs in the disassembly of G4s and iMs during DNA replication and other DNA transactions.

Expression and characterization of pantothenate energy-coupling factor transporters as an anti-infective drug target.

This study investigates the potential of energy-coupling factor (ECF) transporters as promising anti-infective targets to combat antimicrobial resistance (AMR). ECF transporters, a subclass of ATP-binding cassette (ABC) transporters, facilitate the uptake of B-vitamins across bacterial membranes by utilizing ATP as an energy source. Vitamins are essential cofactors for bacterial metabolism and growth, and they can either be synthesized de novo or absorbed from the environment. These transporters are considered promising drug targets, underscoring the need for further research to harness their medicinal potential and develop selective inhibitors that block vitamin uptake in bacteria. Herein, we focused on the ECF transporter for pantothenate (vitamin B5) from Streptococcus pneumoniae and the ECF transporter for folate (vitamin B9) from Lactobacillus delbrueckii as a reference protein. We also included the energizing module for pantothenate along with both full transporter complexes. Initially, we transformed and purified the transporters, followed by an assessment of their thermal stability under various buffer composition, pH, and salt concentrations. Additionally, we monitored the melting temperature over six days to confirm their stability for further assays. We then measured the binding affinities of six ECF inhibitors using surface plasmon resonance (SPR) and evaluated their inhibitory effects through in vitro assays, including bacterial growth assay, whole-cell uptake, and transport-activity assays. After determining cytotoxicity in two human cell lines, we established an in vivo infection model using Galleria mellonella larvae to further validate our findings.

Shape Analysis of Biomimetic and Plasma Membrane Vesicles

Giant membrane vesicles (GUVs) and Giant plasma membrane vesicles (GPMVs) are used as models to study membrane properties. We conducted a comparative study to examine how reducing the volume of vesicles with different lipid compositions, solution symmetries, solution asymmetries, and membrane charges affects their morphology. We used three‐dimensional visualization techniques to study the shape of the vesicles. Although the vesicles may not be perfectly spherical, they exhibit some fluctuations in their shape. To understand these variations, we used confocal image stacks for visualization. Our experimental observations show that the membrane's charge influences the deflation of the GUVs in the presence of trans‐bilayer sugar asymmetries. The lipid bilayers of our GUVs have a uniform distribution of lipids in both leaflets, indicating no asymmetry in lipid composition. However, we induce trans‐bilayer asymmetries by exposing each leaflet to different solution compositions, leading to asymmetric adsorption or desorption layers. We also estimated and compared the deformation of GPMV shapes extracted from cells with trans‐bilayer buffer asymmetries and composition asymmetries with biomimetic membranes.

A Straightforward Model for Quantifying Local pH Gradients Governing the Oxygen Evolution Reaction.

The production and consumption of protons by an electrocatalyst will, under certain conditions, generate localized microenvironments with properties distinct from those of the bulk solution. These local properties are particularly impactful for reactions involving proton-coupled electron transfer, where the generation of locally basic or acidic environments may significantly influence the energy efficiency and reaction selectivity of the electrocatalyst. Whereas local pH environments have been observed and characterized in reductive half-reactions, including the CO2 reduction and hydrogen evolution reactions, the incompatibility of conventional techniques and materials has limited studies in oxidative half-reactions, including the oxygen evolution reaction (OER), which provides the reducing equivalents for solar-to-fuels electrolysis. With the straightforward parameters bulk pH, buffer composition and pKa, and mass transport, we develop a model for describing local pH as a function of current density regardless of the microscopic details of the mechanism. Using an acid-stable PbOx OER catalyst, we observe the formation and dissipation of pH gradients during the OER and validate the model with voltammetric and potentiometric studies. The model predicts how local acidic environments can develop over a narrow OER current density window, thus providing further motivation for the development of OER catalysts that are stable to acid, even when operating in basic aqueous conditions. More generally, the model is not restricted to the OER and is useful for determining the onset of local pH gradients for other electrocatalytic reactions that involve the consumption or generation of protons in energy conversion reactions.

Comparative Assessment of Mitochondria Isolation Buffers for Optimizing Tissue-Specific Yields in Buffalo

Introduction: Mitochondrial studies are crucial for assessing livestock health and performance. While extensive research has been done on cattle and pigs, the influence of mitochondria in Indian buffalo remains unexplored. Therefore, in order to understand functions of mitochondria, their energy-related processes, or any additional mitochondrial traits in buffaloes, it is imperative to isolate high-yield mitochondria with purity and functionality. Mitochondria are extracted by few conventional buffers. These buffers were previously characterized for their effectiveness in isolating mitochondria from rodent and human tissues. Therefore, the present study is to assess the performance of mitochondria isolation buffers specifically in buffalo tissues. Methods: The study involved isolation of mitochondria from four different tissues, i.e., liver, brain, heart and muscles of slaughtered buffalo (n = 3), using: (i) Tris-Mannitol buffer (ii) Tris-Sucrose buffer, and (iii) MOPS-Sucrose buffer. Buffer efficiency in preserving high fidelity during mitochondria isolation was assessed by comparison with Cayman’s MitoCheck® Mitochondrial Isolation Kit (control). Further mitochondrial purity and functionality was assessed through comparative estimation of protein concentration and marker enzyme assays, respectively. Results: Our results revealed insights into the suitability of specific buffer for functional mitochondria isolation from specific type of buffalo tissue. Notably for obtaining high quality functional mitochondria from buffalo, MOPS-Sucrose buffer appeared optimal for soft tissues (liver and brain), while Tris-Mannitol buffer was efficient for hard tissues (muscles and heart). Conclusions: Thus, our research highlights the influence of buffer composition and tissue-specific variations in buffer effectiveness on mitochondrial activity in different tissues, leading to improved mitochondrial isolation in buffalo.

Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. To enhance the signal-to-background ratio of the immunoassay, a GUS switch mutant that exhibits reduced background activation was developed by screening several additional mutations at the diagonal interface residue H514. Moreover, we optimized the composition of the reaction buffer, including organic solvents, salt, and surfactant. Under optimal conditions, we customized OpenGUS immunoassays for Cry j 1, human C-reactive protein, and human lactoferrin, achieving around 10–20-fold maximum fluorescence (15 min) or colorimetric (2 h) responses with picomolar to low nanomolar level detection limit, simply by using commercially available IgGs. Additionally, the three analytes were successfully detected in complex matrices similar to those used in practical applications. We believe that this customizable OpenGUS immunoassay will pave the way for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and onsite environmental assessments.

322 Nutrient transport across gastrointestinal tissues using Ussing chambers

Abstract One of the principal roles of the gut is the absorption of nutrients to meet animal needs. While in vivo measures such as total tract digestibility indicate the totality of the nutrients that disappear, these methods provide no information on the digestion, site, flux, and mechanism of nutrient absorption. Nutrient absorption is a complex set of transport pathways including passive diffusion, facilitated transport, ion exchange, active transport, and secondary active transport that is driven by electrochemical gradients. To tease apart the complexities of nutrient transport at different sites along the gastrointestinal tract, Ussing chambers have been a useful tool. Two ex vivo chambers, filled with oxygenated physiological buffer, and separated by epithelial tissue allow for the detection of nutrient flux, transepithelial currents, and/or membrane permeability. Historically, detection of the nutrient of interest was achieved using radio-labelled or fluorescent tracers. Beyond detecting flux (uptake, efflux, net flux), Ussing chambers can also tease apart the complex network of transport pathways that govern nutrient absorption. Isolating specific transport pathways can be broad spectrum inhibitors, and alterations to the physiological buffers to target transport pathways of interest. Altering the electrical gradient using a voltage clamp for instance, can be used to neutralize, inhibit, or promote, the electrical gradient; such alterations can be especially useful for charged nutrients. Inhibitors, meanwhile, inhibit the activity of entire families of proteins, useful for when the role of specific protein transporters is of interest. Alterations to buffer composition, such as bicarbonate-free buffer solutions or the inclusion and exclusion of compounds with similar size and charge, can isolate transport solely dependent on that solute or provide knowledge on the solutes that may share a common transport mechanism. Together, these modulations allow for a powerful tool to study the presence or absence of nutrient transport and has been crucial in establishing absorption kinetics of ionic nutrients (e.g., Na+, Cl-), small organic nutrients (e.g., short chain fatty acids, glucose), and macromolecules (e.g., immunoglobulins). Done in conjunction with other techniques, Ussing chambers can offer strong insight to improve our understanding of nutrient absorption and epithelial physiology of the gut. This presentation will discuss the methods key to successful use of Ussing chambers, alongside with their limitations and ways to augment data generated by Ussing chambers.

Comparative Analysis of QCM and Electrochemical Aptasensors for SARS-CoV-2 Detection.

The rapid and accurate detection of SARS-CoV-2, particularly its spike receptor-binding domain (S-RBD), was crucial for managing the COVID-19 pandemic. This study presents the development and optimization of two types of aptasensors: quartz crystal microbalance (QCM) and electrochemical sensors, both employing thiol-modified DNA aptamers for S-RBD detection. The QCM aptasensor demonstrated exceptional sensitivity, achieved by optimizing aptamer concentration, buffer composition, and pre-treatment conditions, with a limit of detection (LOD) of 0.07 pg/mL and a linear range from 1 pg/mL to 0.1 µg/mL, and a significant frequency change was observed upon target binding. The electrochemical aptasensor, designed for rapid and efficient preparation, utilized a one-step modification process that reduced the preparation time to 2 h while maintaining high sensitivity and specificity. Electrochemical impedance spectroscopy (EIS) enabled the detection of S-RBD concentrations as low as 132 ng/mL. Both sensors exhibited high specificity, with negligible non-specific interactions observed in the presence of competing proteins. Additionally, the QCM aptasensor's functionality and stability were verified in biological fluids, indicating its potential for real-world applications. This study highlights the comparative advantages of QCM and electrochemical aptasensors in terms of preparation time, sensitivity, and specificity, offering valuable insights for the development of rapid, sensitive, and specific diagnostic tools for the detection of SARS-CoV-2 and other viruses.

Alternative buffer systems in biopharmaceutical formulations and their effect on protein stability.

The formulation of biopharmaceutical drugs is designed to eliminate chemical instabilities, increase conformational and colloidal stability of proteins, and optimize interfacial stability. Among the various excipients involved, buffer composition plays a pivotal role. However, conventional buffers like histidine and phosphate buffers may not always be the optimal choice for all monoclonal antibodies (mAbs). In this study, we investigated the effects of several alternative buffer systems on seven different mAbs, exploring various combinations of ionic strengths, concentrations of the main buffer component, mAb concentrations, and stress conditions. Protein stability was assessed by analyzing soluble aggregate formation through size exclusion chromatography. At low protein concentrations, protein instability after temperature stress was exclusively observed in the bis-TRIS/ glucuronate buffer. Conversely, freeze-thaw stress led to a significant increase in aggregate formation in tested formulations, highlighting the efficacy of several alternative buffers, particularly arginine/ citrate, in preserving protein stability. Under temperature stress, the introduction of arginine to histidine buffer systems provided additional stabilization, while the addition of lysine resulted in protein destabilization. Similarly, the incorporation of arginine into histi-dine/HCl buffer further enhanced protein stability during freeze--thaw cycles. At high protein concentrations, the histidine/citrate buffer emerged as one of the most optimal choices for addressing temperature and light-induced stress. The efficacy of histidine buffers in combating light stress might be attributed to the light-absorbing properties of histidine molecules. Our findings demonstrate that the development of biopharmaceutical formulations should not be confined to conventional buffer systems, as numerous alternative options exhibit comparable or even superior performance.

Feasibility Study for the Use of Gene Electrotransfer and Cell Electrofusion as a Single-Step Technique for the Generation of Activated Cancer Cell Vaccines

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed.Graphical

A combinatorial approach to validate the surface plasmon resonance (SPR) biosensor response

Abstract One of the fundamental challenges of working with surface plasmon resonance (SPR) biosensors is their inherent lack of specificity. Being very sensitive to minute refractive index (RI) changes in their surrounding medium, SPR biosensors are highly susceptible to variations in pH, temperature, and buffer composition. Therefore, it is often necessary to include an additional validation step downstream to SPR biosensing, particularly for clinical analysis. In this proof-of-study work, we have tried to evaluate the utility of surface-enhanced Raman scattering (SERS) tags as secondary labels for validating SPR biosensor response. Accordingly, a Fibre-optic SPR (FO-SPR) biosensor set-up was fabricated by immobilizing anti-BSA antibodies on the sensor platform for capturing and sensing biotinylated-BSA as a model analyte. Subsequently, the bound analyte and the concomitant shift in SPR response were validated by employing streptavidin-functionalized SERS tags. Intriguingly, apart from validation of the SPR response, the SERS tags also significantly improved the sensitivity of the SPR response and provided semi-quantitative information on the bound analyte. Although utilizing SERS tags undermines the label-free tag of SPR biosensors, the huge improvement in sensitivity and specificity of the sensor makes it suitable for clinical analysis. Furthermore, SERS measurements with a portable Raman spectrometer utilized in this study further highlight the potential of this approach for achieving point-of-care (POC) sensing.

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and β-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

An efficient, amine-specific iTRAQ labeling method improves the peptide and protein identification rates

Isotope tags for relative and absolute quantification (iTRAQ) are among the most widely used proteomics quantification techniques. These tags can be rapidly coupled to the primary amines of proteins/peptides through chemical reactions under mild conditions, making this technique universally applicable to any kind of sample. However, iTRAQ reagents also partially react with the hydroxyl groups of serine, threonine and tyrosine residues, particularly when these residues coexist with a histidine residue in the same peptide. This overlabeling of peptides causes systematic biases and significantly compromises protein/peptide identification rates. In this study, we report a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high amine labeling efficiency. The impacts of reaction temperature, reactant concentrations, reaction time, buffer compositions, and pH on iTRAQ labeling performance were investigated in-depth. In a comparison experiment between our method and the standard labeling method provided by the iTRAQ manufacturer, our method reduced the number of overlabeled peptides by 55-fold while achieving comparable amine labeling efficiency. This improvement allowed our method to eliminates the systematic bias against histidyl- and hydroxyl-containing peptides, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins. SignificanceIn addition to amines, the hydroxyl groups in serine, threonine, and tyrosine residues can also partially labeled by iTRAQ reagents, which leads to systematic biases and significantly compromises the analytical sensitivity. To address this issue, we developed a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high labeling efficiency of amines. When benchmarking our method against the standard method provided by the reagent manufacturer, our method achieved comparable labeling efficiency but reduced the overlabeled species by 55-fold. This significant improvement eliminated the systematic biases, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins, demonstrating its superior performance and potential to enhance proteome quantification using iTRAQ labeling.

CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches.

Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air-water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques' abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors.

Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.