Sample Preparation Strategies Research Articles

Influence of different sample preparation approaches on proteoform identification by top-down proteomics.

Top-down proteomics using mass spectrometry facilitates the identification of intact proteoforms, that is, all molecular forms of proteins. Multiple past advances have lead to the development of numerous sample preparation workflows. Here we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified (for example, their number, confidence, physicochemical properties and artificially generated modifications). The various sample preparation strategies resulted in complementary identifications, substantially increasing the proteome coverage. Overall, we identified 13,975 proteoforms from 2,720 proteins of human Caco-2 cells. The results presented can serve as suggestions for designing and adapting top-down proteomics sample preparation strategies to particular research questions. Moreover, we expect that the sampling bias and modifications identified at the intact protein level will also be useful in improving bottom-up proteomics approaches.

Generational Diet-Induced Obesity Remodels the Omental Adipose Proteome in Female Mice.

Obesity, a complex condition that involves genetic, environmental, and behavioral factors, is a non-infectious pandemic that affects over 650 million adults worldwide with a rapidly growing prevalence. A major contributor is the consumption of high-fat diets, an increasingly common feature of modern diets. Maternal obesity results in an increased risk of offspring developing obesity and related health problems; however, the impact of maternal diet on the adipose tissue composition of offspring has not been evaluated. Here, we designed a generational diet-induced obesity study in female C57BL/6 mice that included maternal cohorts and their female offspring fed either a control diet (10% fat) or a high-fat diet (45% fat) and examined the visceral adipose proteome. Solubilizing proteins from adipose tissue is challenging due to the need for high concentrations of detergents; however, the use of a detergent-compatible sample preparation strategy based on suspension trapping (S-Trap) enabled label-free quantitative bottom-up analysis of the adipose proteome. We identified differentially expressed proteins related to lipid metabolism, inflammatory disease, immune response, and cancer, providing valuable molecular-level insight into how maternal obesity impacts the health of offspring. Data are available via ProteomeXchange with the identifier PXD042092.

A 96-position platform for magnetic sorbent-based dispersive microextraction: Application to the determination of tetrahydrocannabinol and cannabidiol in saliva of cannabis smokers

BackgroundIn order to obtain sustainable analytical methods, it is essential to develop kinetically efficient sample preparation strategies in which equilibria are reached faster, as dispersive extraction techniques. In addition, the higher the reduction in size, the higher number of extraction vessels can be located and thus the higher number of samples can be simultaneously treated. All this increases sample throughput, and contributes to the reduction of chemical waste, and energy and sample consumption. In this sense, multiposition extraction platforms are smart strategies to achieve these goals, but they are scarcely developed for dispersive extraction techniques. ResultsTaking miniaturized stir bar sorptive dispersive microextraction (mSBSDME) as a starting point, a 96-position extraction platform has been developed using a 96-position stirring plate and a tailor-designed 3D-printed support for locating the miniaturized extraction vessels, achieving a high-throughput miniaturized sample preparation strategy. In order to show the applicability of this novel platform, the determination of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in human saliva has been carried out and applied to samples collected after the consumption of marijuana and legal CBD-rich cannabis. Only 100 μL of saliva were needed for the analysis and good analytical features in terms of linearity (at least up to 500 ng mL−1), limits of detection (0.7 and 2.8 ng mL−1 for THC and CBD, respectively), and precision (RSD ≤ 14 %) were achieved. SignificanceThe miniaturization of the vessel allows the use of small volumes of sample (i.e., a few microliters) and the treatment of 96 samples in parallel, being the first proposal for carrying out dispersive sorbent-based microextraction under the concept of 96-well format. Additionally, this new workflow contributes to the development of analytical methods that meet the three pillars of sustainability, i.e., greenness and easily affordable in terms of economics and applicability.

Exploring the analytical potential of total reflection X-ray fluorescence (TXRF) combined with partial least square (PLS) for simple determination of ash content in various coal types

Ash content, as a crucial indicator of coal quality, its rapid and accurate determination is pivotal to improve the energy utilization of coal and reduce environmental pollution. Traditional spectroscopic methods face significant challenges in acquiring accurate information from coal samples due to the notorious matrix effects arising from their complex composition, vast molecular structure, and diverse coal types. In this study, the feasibility of total reflection X-ray fluorescence (TXRF) combined with partial least squares (PLS) for the determination of coal ash was firstly investigated based on the TXRF being unaffected by matrix effects. Firstly, coal samples were prepared as suspensions, and the effects of sample particle size and different dispersants on the results of TXRF analyses were evaluated. The accuracy and applicability of the chosen sample preparation strategies were further validated using inductively coupled plasma mass spectrometry (ICP-MS) and two certified reference materials (CRMs). Subsequently, based on the analysis of 19 coal samples, the impact of three different predictive variables on the performance of the PLS model was investigated: (a) TXRF full spectrum normalized by the net intensity of the internal standard; (b) net intensity of characteristic peaks for 12 elements (Al, Si, K, Ca, Ti, Fe, Cr, Mn, Cu, Ni, and Sr) normalized by the net intensity of the internal standard; (c) concentrations of the aforementioned 12 elements. The results demonstrate that the PLS model constructed usingthe TXRF full spectrum normalized by the net intensity of the internal standard exhibits the best predictive capabilities, with the determination coefficient of calibration set (R2) and mean square error (MSE) of the prediction set reaching 0.9736 and 0.99 %, respectively. Moreover, the measurement accuracy of this model was six times greater than that obtained with traditional X-ray fluorescence (XRF). Presented analytical results display the possibilities of combining TXRF with PLS for coal quality evaluation.

Titanium alloys with a high β stabilizer content – sample preparation strategies and micrographs

Abstract Owing to their specific property profile, titanium materials are particularly used in aerospace and medical technology applications. Highly β-stabilized titanium alloys are becoming more and more important for they can be strengthened by martensite decomposition and/or precipitation hardening, thus allowing the property profile to be adapted to the respective application. Not only α and β phase can occur in the respective alloys, but also the α’, α” or ω phase. The sample preparation is particularly problematic when high fractions of α” and β phase are present. They are characterized by a very low stiffness and strength, thus tending to form smear and deformation layers. Three examples will be quoted to describe the sample preparation of titanium materials with a high proportion of β-stabilizers (niobium, molybdenum, and tantalum) and various micrographs will be shown.

Advanced Characterization and Sample Preparation Strategies for Nanoformulations

The escalating impact and remarkable progress of nanotechnology have shifted the paradigms of medicine and the healthcare system. Nanosystems have emerged, extensively holding the potential to advance disease diagnosis and treatment specificity. The extraordinary attributes imparted by nano-systems have helped in overcoming the limitations of conventional interventions to an extent and led to targeted therapy, to name one. The role of nanotechnology in diagnosis is another breakthrough in its appellation. This article aims to address the current characterization and sample preparation techniques for the analysis of nanosystems and provide insights into novel methodologies and in situ instrumentation that have eased sampling procedures.

Recent advances and applications in drug analysis by nano-scale separation techniques

Nano-scale separation techniques (NSTs) offer significant advantages in relation to drug analysis in a wide range of samples. NSTs, including low flow rate LC systems or capillary or chip based electrophoresis/electrochromatography systems, have become the primary tool for advanced drug analysis, and indispensable technology for sensitive and selective drug analysis. In recent decades, significant advances have been achieved using NSTs for drug analysis. In this review, sample preparation strategies, new advances and applications in NSTs and the contribution toward forensic science applications were reported. In addition, some recent and selected applications with or without mass spectrometry (e.g., low resolution/high resolution -MS) are summarized.

Pinpointing the microbiota of tardigrades: What is really there?

Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets. This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Comparison of the Sample Preparation Strategies and Impacts on the Tensile Strength of Gas Shale with Variable Moisture Conditions

Moisture significantly affects the mechanical behavior of gas shale and further determines the hydraulic fracturing performance, as it is more attractive. Nevertheless, batch experiments have usually involved variable methodologies regarding the preparation of moisture-contained shale specimens in the sequence (and/or frequency) of drying and soaking treatments. Accordingly, this work investigates how the preparation methodology influences the test results of moisture-contained shale samples. This study compares three commonly used shale sample preparation strategies for acquiring different moisture contents, that is, “dry-wet”, “dry-wet-dry”, and “wet-dry-wet” strategies, followed by a Brazilian splitting test for the mechanical parameters. The results show that under the same saturation conditions, the longer the soaking time during sample preparation, the higher the degradation degree of shale tensile strength. Meanwhile, prolonged soaking can lead to a more discrete distribution of strength values, and the failure mode may deviate from the Brazilian splitting theory model. Under the combined influence of moisture content and soaking time, the tensile strength of shale decreases approximately linearly with increasing saturation, while the degradation degree increases nonlinearly with increasing saturation, and the degradation rate changes from slow to fast. According to the observation of the microstructure of hydrated shale, prolonged soaking can lead to an increase in the expansion of clay minerals in shale by hydration, resulting in looser and more fragmented internal structure, and further degradation in shale strength. In order to weaken the interference of hydration when studying the effect of moisture content on the tensile strength of shale, the soaking time should be minimized as much as possible during the preparation process.

Trace analysis of 20 antihistamines in milk by ultrahigh performance liquid chromatography coupled with high field quadrupole orbitrap high resolution mass spectrometry followed dispersive micro solid phase extraction

Ultrahigh-performance liquid chromatography coupled with high-field quadrupole Orbitrap high resolution mass spectrometry was used for the separation and determination of 20 antihistamines, and a dispersive micro solid-phase extraction procedure using high-performance absorbing material was developed as a sample preparation strategy for extracting 20 antihistamines from milk. Instrument conditions and key parameters influencing extraction efficiency were investigated to obtain an optimized method. The limit of detection for 20 antihistamines in milk using this method is 0.05 µg/L to 1.0 µg/L. Recoveries are between 80.7 % and 108.3 %, and the relative standard deviation is less than 15 %. It is suitable for confirmatory monitoring and quantitative analysis of 20 antihistamines in milk. The results show that antihistamines in milk may be noteworthy issues for human health and environmental pollution.

Room-temperature fabrication of fluorinated covalent organic polymer @ Attapulgite composite for in-syringe membrane solid-phase extraction and analysis of domoic acid in aquatic products

A novel fluorinated covalent organic polymer @ attapulgite composite (F-COP@ATP) was prepared at room temperature for in-syringe membrane solid-phase extraction (SM-SPE) of domoic acid (DA) in aquatic products. Natural ore ATP has the advantages of low cost, good mechanical strength and abundant hydroxyl group on its surface, and in-situ modified F-COP layer can provide abundant adsorption sites. F-COP@ATP combining the advantages of F-COP and ATP, becomes an ideal adsorbent for DA extracting. Moreover, a high-throughput sample preparation strategy was carried out by using the F-COP@ATP membrane as syringe filter and assembling syringes with a ten-channel injection pump. In addition, the experimental factors were optimized, such as pH of extract, amount of adsorbent, velocity of extraction and desorption, type and volume of desorption solvent. The DA analytical method was established by SM-SPE-HPLC/tandem mass spectrometry. The method had a wide linear range with low limit of detection (0.344 ng/kg) and low limit of quantification (1.14 ng/kg). F-COP@ATP membrane can be reused more than five times. The method realized the analysis of DA in scallop and razor clam samples, which shows its application prospect in practical analysis. This study provided an efficient, low-energy and mild idea for preparing other reusable natural mineral ATP-based composite materials for separation and enrichment, which reduces the experimental cost and is closer to environmental protection and green chemistry to a certain extent.

Microfluidic Impedance Cytometry Enabled One-Step Sample Preparation for Efficient Single-Cell Mass Spectrometry.

Single-cell mass spectrometry (MS) is significant in biochemical analysis and holds great potential in biomedical applications. Efficient sample preparation like sorting (i.e., separating target cells from the mixed population) and desalting (i.e., moving the cells off non-volatile salt solution) is urgently required in single-cell MS. However, traditional sample preparation methods suffer from complicated operation with various apparatus, or insufficient performance. Herein, a one-step sample preparation strategy by leveraging label-free impedance flow cytometry (IFC) based microfluidics is proposed. Specifically, the IFC framework to characterize and sort single-cells is adopted. Simultaneously with sorting, the target cell is transferred from the local high-salinity buffer to the MS-compatible solution. In this way, one-step sorting and desalting are achieved and the collected cells can be directly fed for MS analysis. A high sorting efficiency (>99%), cancer cell purity (≈87%), and desalting efficiency (>99%), and the whole workflow of impedance-based separation and MS analysis of normal cells (MCF-10A) and cancer cells (MDA-MB-468) are verified. As a standalone sample preparation module, the microfluidic chip is compatible with a variety of MS analysis methods, and envisioned to provide a new paradigm in efficient MS sample preparation, and further in multi-modal (i.e., electrical and metabolic) characterization of single-cells.

An innovative sample preparation strategy to visualize the crystalline structure of fiber reinforced thermoplastic composites by polarized optical microscopy

AbstractThis study aims at an innovative sample preparation strategy for fiber reinforced thermoplastic (FRTP) composites analyzed via polarized optical microscopy to visualize the crystalline structure of the matrix resin. Investigating the structure is relevant for understanding the relationship with the mechanical properties. In general, polarized optical microscopy is a powerful technique for visualizing polymer crystalline structures at samples sliced into thin sections below 30 μm. However, analyzing FRTPs remains challenging in absence of suitable methods for the preparation of thin sections owing to the embedded fibers. Herein, the preparation of appropriately thin sections in a novel sequence of steps is shown that may generically be applied to FRTPs. Four different types of FRTPs with major matrix resins (GF/PBT, CF/PA6, CF/PEEK and GF/PP) were successfully processed to thickness of 5 μm or below without fiber loss, resin breakage or delamination between fiber and resin demonstrating the utility of the developed method, which is the significant outcome of the study. To the best of our knowledge, the subsequently obtained polarized optical microscopic images for the first time visualized the crystalline structure of the matrix resin revealing nucleation of crystals from the fiber surface and effects of mold flow on the crystallization behavior.Highlights Thinning of fiber reinforced thermoplastics to thickness of 5 μm or below. Thinning achieved despite fiber volume at industrial applications level. Sample preparation ensures ideal conditions for polarized optical microscopy. Visualization of crystalline structure of the matrix resin for the first time. Potential advancement for spectroscopic and electron microscopic analyses.

A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis.

Bottom-up proteomic approaches depend on the efficient digestion of proteins into peptides for mass spectrometric analysis. Sample preparation strategies, based on magnetic beads, filter-aided systems, or in-solution digests, are commonly used for proteomic analysis. Time-intensive methods like filter-aided sample preparation (FASP) have led to the development of new, more time-efficient filter-based strategies like suspension trappings (S-Traps) or magnetic bead-based strategies like SP3. S-Traps have been reported as an alternative proteomic sample preparation method as they allow for high sodium dodecyl sulfate (SDS) concentrations to be present in the sample. In this study, we compare the efficiency of different protocols for FASP, SP3, and S-Trap-based digestion of proteins after extraction from Trichomonas vaginalis. Overall, we found a high number of protein IDs for all tested methods and a high degree of reproducibility within each method type. However, FASP with a 3 kDa cutoff filter unit outperformed the other methods analyzed, referring to the number of protein IDs. This is the first work providing the direct comparison of four different bottom-up proteomic approaches regarding the most efficient proteomic sample preparation protocol for the human parasite T. vaginalis.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

Targeted Phosphoproteomics of Human Saliva Extracellular Vesicles via Multiple Reaction Monitoring Cubed (MRM3).

Oral squamous cell carcinoma (OSCC) has become a global health problem due to its increasing incidence and high mortality rate. Early intervention through monitoring of the diagnostic biomarker levels during OSCC treatment is critical. Extracellular vesicles (EVs) are emerging surrogates in intercellular communication through transporting biomolecule cargo and have recently been identified as a potential source of biomarkers such as phosphoproteins for many diseases. Here, we developed a multiple reaction monitoring cubed (MRM3) method coupled with a novel sample preparation strategy, extracellular vesicles to phosphoproteins (EVTOP), to quantify phosphoproteins using a minimal amount of saliva (50 μL) samples from OSCC patients with high specificity and sensitivity. Our results established differential patterns in the phosphopeptide content of healthy, presurgery, and postsurgery OSCC patient groups. Notably, we discovered significantly increased salivary phosphorylated alpha-amylase (AMY) in the postsurgery group compared to the presurgery group. We hereby present the first targeted MS method with extremely high sensitivity for measuring endogenous phosphoproteins in human saliva EVs.

Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis

Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis performances are significantly influenced by the chemical composition and the complexity of the sample. In various applications, that impact has prevented the use of CE-MS for the characterization and quantification of proteins in biological samples. Here we present the development and evaluation and a sample preparation procedure, based on affinity purification, for the specific extraction of the monoclonal antibody (mAbs) infliximab from human serum in order to perform subsequent proteolytic digestion and CE-MS/MS analysis. Three distinctive sample preparation strategies were envisaged. In each case, the different steps composing the protocol were thoroughly optimized and evaluated in order to provide a sample preparation addressing the important complexity of serums samples while providing an optimal compatibility with CE-MS/MS analysis. The different sample preparation strategies were assessed concerning the possibility to achieve an appropriate absolute quantification of the mAbs using CE-MS/MS for samples mimicking patient serum samples. Also, the possibility to perform the characterization of several types of post-translational modifications (PTMs) was evaluated. The sample preparation protocols allowed the quantification of the mAbs in serums samples for concentration as low as 0.2 µg·mL−1 (2.03 nM) using CE-MS/MS analysis, also the possibility to characterize and estimate the modification level of PTMs hotspots in a consistent manner. Results allowed to attribute the effect on the electrophoretic separation of the different steps composing sample preparation. Finally, they demonstrated that sample preparation for CE-MS/MS analysis could benefit greatly for the extended applicability of this type of analysis for complex biological matrices.

Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel "direct" single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.

Совершенствование схем анализа материалов на основе карбида вольфрама

Using atomic emission spectrometry with inductively coupled plasma and flame version of atomic absorption spectrometry, methods for determining the content of Co, Cr, Ti, V in a wide concentration range from 1·10–3 to n·10 % in tungsten carbide-based materials without matrix separation and using certified solid standard samples with good metrological characteristics were developed. The relative standard deviation is 0.05 – 0.005 when the content of elements is from 1 to 20 % and does not exceed 0.15 when the content of elements is from 0.001 to 0.1 %. Depending on the chemical, phase and disperse composition, as well as the method of obtaining tungsten carbide-based compounds that form different types of materials, which are very different in their ability to dissolve, various sample preparation schemes were used. The analytical parameters for the determination of Co, Cr, Ti, V were found, the operating conditions of the spectrometers were optimized, and calibration methods were selected. The influence of the tungsten matrix element on the analytical signals of analytes and ways of taking it into account have been studied. The possibilities of using spectroscopic methods for the determination of Co, Cr, Ti, V are compared. It is shown that the ICP AES method is dominant in the determination of elements in the concentration range from 1·10–3 to n·10 % in materials based on tungsten carbide. The proposed methods of analytical control will provide research on the creation of new materials based on tungsten carbide, including nanostructured ones, with extremely high physical and mechanical properties, which is especially important in the context of import substitution of materials.