Sample Preparation Strategies Research Articles

Strategies in the analysis of samples of composites for the quantification of porosity by image analysis from optical microscopy

Porosity and voids are a type of defect that may appear during the manufacturing of composite structures. Its possible presence is checked in production parts by different non-destructive techniques based on ultrasonic inspection. It is important to perform this evaluation  considering that a high content of these defects decrease the mechanical properties compared to the original design. In recent years, optical microscopy of composite materials has become a very powerful tool, which is  available for the industry to inspect and look at the internal structure and help to reach conclusions in the field of quality, failure analysis and I+D+I. In addition, the use of automatic scanning techniques of wide cross sections has opened the possibility of quantifying the percentage of porosity/voids, as well as become an alternative to more laborious testing procedures such as acid digestion of the resins. This study contributes to existing test standards within the aeronautical sector, in the sample preparation strategy. For this purpose, samples of epoxy matrices with carbon fibers, both unidirectional and fabric tapes have been analyzed to evaluate the influence of the quality of sample preparation, the stacking sequence and the initial porosity levels, the repeatability of the measurements and the quality of the results. An attempt has been made to correlate the results with standar physical / chemical analysis.

Flow Cytometry-Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments.

Since decades, flow cytometry (FC) is a powerful technique to perform single cell analyses with high accuracy and throughput. Moreover, FC is the method of choice to study bacterial cell heterogeneity and complements single-cell imaging techniques. The complex experimental approaches for FC sample preparation and the subsequent FC adjustment and gating strategy demand careful considerations to be successful when analyzing complex microbial populations, especially when liberated populations of intracellular bacterial pathogens, or bacterial pathogens inside intact host cells are analyzed. Here, we provide a set of experimental protocols for FC sample preparation of (1) in vitro cultured bacterial cells, (2) liberated intracellular bacteria from host cells, or (3) preparation of intact infected phagocytic or epithelial cells commonly used as host cells in infection biology. Since sample preparation, cytometer adjustment, and gating strategy are essential for experimental success, we aim to provide our expertise to support application of FC by other researchers.

Microwave-induced combustion for petroleum coke digestion: a promising sample preparation strategy for subsequent elemental determination

The increasing demand for the elemental determination of petrochemical samples by inductively coupled plasma techniques requires the development of suitable sample preparation methods that are in accordance with green chemistry recommendations.

Key Steps to Create a Sample Preparation Strategy for Inductively Coupled Plasma (ICP) or ICP–Mass Spectrometry (ICP-MS) Analysis

Inductively coupled plasma (ICP) is a well-known source for atomic spectroscopy, either using optical emission spectroscopy (OES) or mass spectrometry (MS) as a detection technique. The high energy and temperatures in an ICP (typically between 6,000 to 8,000 °C) lead to an efficient and complete decomposition of the sample as well as the vaporization, excitation, and possibly ionization of atoms in their gas phase. The high temperature of the plasma makes this source inherently robust and tolerant to a wide variety of sample types (such as solutions containing an elevated percentage of dissolved solids, different acids, and organic solvents). However, inadequate sample preparation, or inappropriate configuration of the sample introduction system, can have negative consequences such as drift of the analytical signal, increased backgrounds, inadequate detection limits, or unexpected interferences. This tutorial highlights the most important steps necessary for creating a trouble-free sample preparation workflow for elemental analysis.

A fully integrated sample preparation strategy for highly sensitive intact glycoproteomics.

A fully integrated sample preparation technology, termed Intact GlycoSISPROT, was developed for the highly sensitive analysis of site-specific glycopeptides. Through integrating all glycoproteomic sample preparation steps into a single spintip, Intact GlycoSISPROT provided a tool for site-specific glycosylation analysis with low micrograms to even nanograms of protein sample.

Selective extraction of gambierone and related metabolites in Gambierdiscus silvae using m-aminophenylboronic acid–agarose gel and liquid chromatography–high-resolution mass spectrometric detection

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography–high-resolution mass spectrometry (LC–HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC–HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.

Comprehensive Strategy for Sample Preparation for the Analysis of Food Contaminants and Residues by GC-MS/MS: A Review of Recent Research Trends.

Food safety and quality have been gaining increasing attention in recent years. Gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), a highly sensitive technique, is gradually being preferred to GC–MS in food safety laboratories since it provides a greater degree of separation on contaminants. In the analysis of food contaminants, sample preparation steps are crucial. The extraction of multiple target analytes simultaneously has become a new trend. Thus, multi-residue analytical methods, such as QuEChERs and adsorption extraction, are fast, simple, cheap, effective, robust, and safe. The number of microorganic contaminants has been increasing worldwide in recent years and are considered contaminants of emerging concern. High separation in MS/MS might be, in certain cases, favored to sample preparation selectivity. The ideal sample extraction procedure and purification method should take into account the contaminants of interest. Moreover, these methods should cooperate with high-resolution MS, and other sensitive full scan MSs that can produce a more comprehensive detection of contaminants in foods. In this review, we discuss the most recent trends in preparation methods for highly effective detection and analysis of food contaminants, which can be considered tools in the control of food quality and safety.

Emerging Contributions of Solid-State NMR Spectroscopy to Chromatin Structural Biology.

The eukaryotic genome is packaged into chromatin, a polymer of DNA and histone proteins that regulates gene expression and the spatial organization of nuclear content. The repetitive character of chromatin is diversified into rich layers of complexity that encompass DNA sequence, histone variants and post-translational modifications. Subtle molecular changes in these variables can often lead to global chromatin rearrangements that dictate entire gene programs with far reaching implications for development and disease. Decades of structural biology advances have revealed the complex relationship between chromatin structure, dynamics, interactions, and gene expression. Here, we focus on the emerging contributions of magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS NMR), a relative newcomer on the chromatin structural biology stage. Unique among structural biology techniques, MAS NMR is ideally suited to provide atomic level information regarding both the rigid and dynamic components of this complex and heterogenous biological polymer. In this review, we highlight the advantages MAS NMR can offer to chromatin structural biologists, discuss sample preparation strategies for structural analysis, summarize recent MAS NMR studies of chromatin structure and dynamics, and close by discussing how MAS NMR can be combined with state-of-the-art chemical biology tools to reconstitute and dissect complex chromatin environments.

Recent advances in mass spectrometry analysis of neuropeptides.

Due to their involvement in numerous biochemical pathways, neuropeptides have been the focus of many recent research studies. Unfortunately, classic analytical methods, such as western blots and enzyme-linked immunosorbent assays, are extremely limited in terms of global investigations, leading researchers to search for more advanced techniques capable of probing the entire neuropeptidome of an organism. With recent technological advances, mass spectrometry (MS) has provided methodology to gain global knowledge of a neuropeptidome on a spatial, temporal, and quantitative level. This review will cover key considerations for the analysis of neuropeptides by MS, including sample preparation strategies, instrumental advances for identification, structural characterization, and imaging; insightful functional studies; and newly developed absolute and relative quantitation strategies. While many discoveries have been made with MS, the methodology is still in its infancy. Many of the current challenges and areas that need development will also be highlighted in this review.

Preparation strategy for statistically significant micrometer-sized particle systems suitable for correlative 3D imaging workflows on the example of X-ray microtomography

The characterization of multidimensional particle property distributions through computed tomography requires an adapted sample preparation strategy. This strategy should both generate as many spatially separated particles as possible in the smallest achievable volumes and also enable mechanically and vacuum-stable samples that are suitable for correlative measurement, for example with high-energy ion beam methods. In the present study an epoxy-based method is presented that minimizes the negative influence of particle sedimentation by adding very low X-ray absorbing graphite nanoparticles as spacer. A machine learning-based method is presented to discretize the particle system. Results are compared with data from 2D SEM validation measurements and data of a previous study.

Determination of pesticide residues in wine by solid-phase extraction on-line combined with liquid chromatography tandem mass spectrometry

Liquid chromatography tandem mass spectrometry (LCMS/MS) has been combined with different sample preparation strategies to determine potential pesticide residues existing in wine. Herein, we propose the on-line coupling between solid-phase extraction (SPE), based on a reversed-phase polystyrene-divinyl benzene (PS-DVB) sorbent, and LCMS/MS for the automated monitoring of a selection of 48 pesticides (37 fungicides and 11 insecticides) in this beverage. Parameters affecting the sensitivity, selectivity, accuracy and robustness of the method are evaluated as function of sample volume, clean-up conditions, and number of extraction-desorption cycles performed with the same SPE cartridge. Considering a sample volume of 3 μL, the on-line SPE LCMS/MS method was free of MEs, attaining LOQs below 2.5 μg L−1, and providing recoveries in the range from 70 to 120 % for 90 % of investigated compounds. Increasing the volume of sample loaded in the on-line SPE cartridge to 25 μL permitted a further reduction of LOQs, with a linear response range (0.1–25 μg L −1), covering the analysis of low residue samples. Twelve out of 48 investigated compounds were noticed at individual concentrations above 10 μg L−1 in at least one out of 30 processed wines. In white wines, the sum of concentrations for iprovalicarb, fenhexamide and metalaxyl represented more than 50 % of total residues. As regards red wines, tebuconazole, cyprodinil and metalaxyl were the predominant species.

Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample’s PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

Applications and continued evolution of glycan imaging mass spectrometry.

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

Simultaneous LC-MS/MS bioanalysis of alkaloids, terpenoids, and flavonoids in rat plasma through salting-out-assisted liquid-liquid extraction after oral administration of extract from Tetradium ruticarpum and Glycyrrhiza uralensis: a sample preparation strategy to broaden analyte coverage of herbal medicines.

Herbal medicines have historically been practiced in combinatorial way, which achieves therapeutic efficacy by integrative effects of multi-components. Thus, the accurate and precise measurement of multi bioactive components in matrices is inalienable to understanding the metabolism and disposition of herbal medicines. In this study, aiming to provide a strategy that improves analyte coverage, evaluation of six protocols employing sample pretreatment methods- protein precipitation (PPT), liquid-liquid extraction (LLE), sugaring-out-assisted liquid-liquid extraction (SULLE), and salting-out-assisted liquid-liquid extraction (SALLE)- was performed by LC-MS/MS using rat plasma and a mixture of alkaloid (evodiamine, rutaecarpine, dehydroevodiamine), terpenoid (limonin, rutaevin, obacunone), and flavonoid (liquiritin, isoliquiritin, liquiritigenin) standards isolated from Tetradium ruticarpum and Glycyrrhiza uralensis. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) LLE with methyl tertiary-butyl ether-dichloromethane, (4) LLE with ethyl acetate-n-butanol, (5) SALLE with ammonium acetate, (6) SULLE with glucose. The results suggested that SALLE produced broader analyte coverage with satisfactory reproducibility, acceptable recovery, and low matrix interference. Then, sample preparation procedure of SALLE, chromatographic conditions, and mass spectrometric parameters were optimized, followed by method validation, showing that good sensitivity (LLOQ ≤1 ng mL-1), linearity (r ≥0.9933),precision (RSD≤ 14.45%), accuracy (89.54~110.87%), and stability could be achieved. Next, the developed method was applied successfully to determine the pharmacokinetic behavior of the nine compounds in rat plasma after intragastric administration with an extract from Tetradium ruticarpum and Glycyrrhiza uralensis (Wuzhuyu-Gancao pair). Based on an extensive review and experiments, a sample preparation procedure that matches with LC-MS/MS technique and can get wider analyte coverage was outlined. The developed SALLE method is rapid, reliable, and suitable for bioanalysis of analytes with diverse polarity, which was expected to be a promising strategy for the pharmacokinetic studies of herbal medicines. Graphical abstract.

Evaluation of measurable residual disease in multiple myeloma by multiparametric flow cytometry: Current paradigm, guidelines, and future applications.

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.

Simultaneous determination of organophosphorus pesticides residues in vegetable, fruit juice, and milk samples with magnetic dispersive micro solid-phase extraction and chromatographic method; recruitment of simplex lattice mixture design for optimization of novel sorbent composites

Dispersive micro solid-phase extraction procedure using a novel and selective sorbent prepared from four components was developed as a sample preparation strategy for extracting five organophosphorus pesticides, including fenitrothion, malathion, ethion, and chlorpyrifos, and diazinon in several vegetables, fruit juices, and cow's milk samples. Due to the high importance of the sorbent in the microextraction process, the percentages of sorbent components, including metal-organic framework (ZIF-67), chitosan, magnetic Fe3O4 nanoparticles, and silica nanoparticles, were optimized by a simplex lattice mixture design. After optimizing the sorbent composite, effective parameters on the extraction of organophosphorus pesticides were optimized using a definitive screening design and Box-Behnken design, respectively. A surfactant (Triton X100) as a dispersion agent with a low volume (10 μL) was utilized in the microextraction procedure to reduce the sorbent dispersion time and increase the sorbent dispersion efficiency. Under the optimal conditions, linearity for the determination of fenitrothion, malathion, ethion, chlorpyrifos, and diazinon was in the concentration ranges of 0.13–1100, 0.27–1000, 0.38–1000, 0.21–1200, and 0.11–1100 ng mL−1 with a determination coefficient higher than 0.9906, respectively. The quantitation limits, detection limits, and relative standard deviations (n = 5) were lower than 0.38 ng mL−1, 0.11 ng mL−1, and 4.59% for the determination of organophosphorus pesticides. The method application for measuring OPPs on cucumber, carrot, tomato, apple juice, orange juice, and cow's milk indicated the presence of residual amounts of malathion in a cucumber sample, diazinon in a carrot sample, and chlorpyrifos in a tomato sample.

Quantitative analysis of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells using UHPLC-MS/MS.

γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer in HeLa cells using ultra-high-performance liquid chromatography-tandem mass spectrometry. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells.

Sample Preparation Strategies for Antibody-Free Quantitative Analysis of High Mobility Group Box 1 Protein.

Sickness behavior and fatigue are induced by cerebral mechanisms involving inflammatory cytokines. High mobility group box 1 (HMGB1) is an alarmin, and a potential key player in this process. Reliable quantification methods for total HMGB1 and its redox variants must be established in order to clearly understand how it functions. Current methods pose significant challenges due to interference from other plasma proteins and autoantibodies. We aimed to develop an antibody-free sample preparation method followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma. Different methods were applied for the removal of interfering proteins and the enrichment of HMGB1 from spiked human plasma samples. A comparison of methods showed an overall low extraction recovery (<40%), probably due to the stickiness of HMGB1. Reversed-phase liquid chromatography separation of intact proteins in diluted plasma yielded the most promising results. The method produced an even higher degree of HMGB1 purification than that observed with immunoaffinity extraction. Detection sensitivity needs to be further improved for the measurement of HMGB1 in patient samples. Nevertheless, it has been demonstrated that a versatile and fully antibody-free sample preparation method is possible, which could be of great use in further investigations.

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions.

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membraneinteractions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.