Isolation of extracellular vesicles from human plasma samples: The importance of controls.

BACKGROUND: Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. RATIONAL: Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. METHOD AND RESULTS: We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. CONCLUSION: Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Extraction of Functional Mitochondria Based on Membrane Stiffness.

The abnormal functionality of mitochondria has been linked to many life-threatening diseases such as cancers, failure of cardiovascular functions, and neurodegenerative disorders. Therefore, in vitro analysis of mitochondria has garnered great interest for understanding the mechanism of mitochondrial dysfunction-related disease development and therapeutics. However, due to the intrinsic heterogeneity of cell membrane stiffness, it remains challenging to standardize the protocols for the extraction of mitochondria and adequate disruption of the cellular membrane while retaining the functionality of mitochondria. We have previously developed a microfluidics-based cell shredder capable of serving the purpose. In this protocol, we describe the step-by-step procedures to empirically identify the threshold shear stress using this microfluidics-based cell shredder for mitochondrial extraction. The optimal shear stress to disrupt human embryonic kidney cell (HEK 293) and mice muscle cell (C2C12) has been characterized at around 16.4 Pa, whereas cell lines with stiffer membrane stiffness, for example, neuroblastoma cells (SH-SY5Y), require 27.4 Pa to effectively lyse the cells. This protocol also provides detailed procedures to determine the quality of extracted mitochondria based on the membrane potential and the integrity of extracted mitochondria. A comparison with the widely employed Dounce homogenizer has shown that the proposed microscale cell shredder can yield at least 40% more functional mitochondria and retain higher integrity regarding extracted mitochondria than the counterparts extracted from Dounce homogenizer, especially for low cell concentrations (5-20 × 104 cells/mL) and small sample volume (<200 µL).

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

Elucidating the combined effect of sample preparation and solid-phase microextraction conditions on the volatile composition of cooked meat analyzed by capillary gas chromatography coupled with mass spectrometry.

Solid-phase microextraction coupled to gas chromatography-mass spectrometry is a common approach to analyze the volatile profile of cooked meat. The present study aims to investigate the combined effect of sample preparation, including meat presentation (minced and steak) and cooking method (stewed and grilled), and extraction temperature (30, 60 and 80 °C) and time (30 and 50 min) on the volatile composition of cooked deer meat. The statistical results indicated that extraction temperature was the most relevant factor affecting the meat volatile profile of cooked meat followed by the extraction time. Higher extraction temperatures improved the detection of heavy volatile compounds, while sample preparation had little influence on the meat volatile profile, probably due to the accurate control of the parameters used for meat presentation and cooking methods. The results of this work can assist in the standardization of analytical procedures for the characterization of volatile compounds in cooked meat.

Fabric Phase Sorptive Extraction: A Paradigm Shift Approach in Analytical and Bioanalytical Sample Preparation.

Fabric phase sorptive extraction (FPSE) is an evolutionary sample preparation approach which was introduced in 2014, meeting all green analytical chemistry (GAC) requirements by implementing a natural or synthetic permeable and flexible fabric substrate to host a chemically coated sol-gel organic-inorganic hybrid sorbent in the form of an ultra-thin coating. This construction results in a versatile, fast, and sensitive micro-extraction device. The user-friendly FPSE membrane allows direct extraction of analytes with no sample modification, thus eliminating/minimizing the sample pre-treatment steps, which are not only time consuming, but are also considered the primary source of major analyte loss. Sol-gel sorbent-coated FPSE membranes possess high chemical, solvent, and thermal stability due to the strong covalent bonding between the fabric substrate and the sol-gel sorbent coating. Subsequent to the extraction on FPSE membrane, a wide range of organic solvents can be used in a small volume to exhaustively back-extract the analytes after FPSE process, leading to a high preconcentration factor. In most cases, no solvent evaporation and sample reconstitution are necessary. In addition to the extensive simplification of the sample preparation workflow, FPSE has also innovatively combined the extraction principle of two major, yet competing sample preparation techniques: solid phase extraction (SPE) with its characteristic exhaustive extraction, and solid phase microextraction (SPME) with its characteristic equilibrium driven extraction mechanism. Furthermore, FPSE has offered the most comprehensive cache of sorbent chemistry by successfully combining almost all of the sorbents traditionally used exclusively in either SPE or in SPME. FPSE is the first sample preparation technique to exploit the substrate surface chemistry that complements the overall selectivity and the extraction efficiency of the device. As such, FPSE indeed represents a paradigm shift approach in analytical/bioanalytical sample preparation. Furthermore, an FPSE membrane can be used as an SPME fiber or as an SPE disk for sample preparation, owing to its special geometric advantage. So far, FPSE has overwhelmingly attracted the interest of the separation scientist community, and many analytical scientists have been developing new methodologies by implementing this cutting-edge technique for the extraction and determination of many analytes at their trace and ultra-trace level concentrations in environmental samples as well as in food, pharmaceutical, and biological samples. FPSE offers a total sample preparation solution by providing neutral, cation exchanger, anion exchanger, mixed mode cation exchanger, mixed mode anion exchanger, zwitterionic, and mixed mode zwitterionic sorbents to deal with any analyte regardless of its polarity, ionic state, or the sample matrix where it resides. Herein we present the theoretical background, synthesis, mechanisms of extraction and desorption, the types of sorbents, and the main applications of FPSE so far according to different sample categories, and to briefly show the progress, advantages, and the main principles of the proposed technique.

Development of a sample preparation approach for the analysis of fining agents in wines by liquid chromatography with tandem mass spectrometry.

During winemaking a wide variety of processing aids such as albumin, caseinates and lysozyme are often used. These proteins are considered allergenic and could become a human health risk in susceptible individuals. In our knowledge, there are no methods published for the analysis of these three proteins simultaneously by liquid chromatography with tandem mass spectrometry, with electrospray ionization. Therefore, in this work, a sample preparation approach for the analysis of α-casein, ß-casein, albumin and lysozyme, in a single run, was performed and compared with published data. Through a pH adjustment, combining the use of cellulose ester membranes, a precipitation with organic solvents and a final concentration/clean-up, we achieved recovery values from 90.7 to 108.6%. The method was validated, showing determination coefficients R2 ≥ 0.99. This method was able to quantify proteins even at lower levels (limits of quantification from 0.01 to 0.25 mg/L) than the current legal limits.

"Intensification of Vaporization by Decompression to the Vacuum" (IVDV), a novel technology applied as a pretreatment to improve polyphenols extraction from olive leaves.

Impact of the "Intensification of Vaporization by Decompression to the Vacuum" (IVDV) on extraction of polyphenols from olive leaves was investigated. Using Response Surface Methodology, the effect of three variables were studied: initial water content of leaves, processing time and steam pressure on total phenolic content (TPC). Extractions of TPC from leaves were achieved either using 100% water as a solvent (w100), or 50% (v/v) aqueous ethanol (w50). Following IVDV pretreatment, TPC yields were enhanced with both solvents by approximately 3 times compared to the negative controls. Furthermore, oleuropein and hydroxytyrosol were intensified by up to 600% and 238% respectively. Antioxidant-antiradical assays revealed higher activities, up to 3.5 times, in extracts from IVDV-treated leaves. Calculation of the extraction indices Zp, reflecting cellular damage, confirmed the beneficial effect of IVDV on the extraction yield. Finally, Scanning Electron Microscopy (SEM) permitted the morphological observation of IVDV-treated as compared to untreated leaves.

Experimental and Data Analysis Considerations for Three-Dimensional Mass Spectrometry Imaging in Biomedical Research.

Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.

Features of sample preparation techniques in the total-reflection X-ray fluorescence analysis of tea leaves.

Tea is a popular drink around the world and contains essential minerals and trace elements for human health. In this study, the analytical capabilities of the total-reflection X-ray fluorescence method (TXRF) were considered for the analysis of tea. Different sample preparation techniques, e.g. suspension, open vessel acid digestion, and microwave acid digestion were examined. The influence of particle size was investigated in the analysis of the suspension of tea samples. Mass-absorption coefficients for the tea matrix and the critical surface density of the specimen were calculated. The data obtained explain the presence of absorption effects in the determination of P, S, Cl, and K in suspensions. The digestion procedure is chosen as an optimal sample preparation technique for the TXRF analysis of tea. Nineteen real tea samples were analyzed using TXRF. The advantages of TXRF have been presented through the comparison of results with the wavelength-dispersive X-ray fluorescence spectrometry method.

Monitoring AuNP Dynamics in the Blood of a Single Mouse Using Single Particle Inductively Coupled Plasma Mass Spectrometry with an Ultralow-Volume High-Efficiency Introduction System.

Gold nanoparticles (AuNPs) are increasingly being used as diagnostic and therapeutic agents owing to their excellent properties; however, there is not much data available on their dynamics in vivo on a single particle basis in a single mouse. Here, we developed a method for the direct analysis of nanoparticles in trace blood samples based on single particle inductively coupled plasma-mass spectrometry (spICP-MS). A flexible, highly configurable, and precisely controlled sample introduction system was designed by assembling an ultralow-volume autosampler (flow rate in the range of 5-5000 µL/min) and a customized cyclonic spray chamber (transfer efficiency up to 99%). Upon systematic optimization, the detection limit of the nanoparticle size (LODsize) of AuNPs in ultrapure water was 19 nm, and the detection limit of the nanoparticle number concentration (LODNP) was 8 × 104 particle/L. Using a retro-orbital blood sampling method and subsequent dilution, the system was successfully applied to track the dynamic changes in size and concentration for AuNPs in the blood of a single mouse, and the recovery for the blood sample was 111.74%. Furthermore, the concentration of AuNPs in mouse blood reached a peak in a short period of time and, then, gradually decreased. This study provides a promising technique for analyzing and monitoring the size and concentration of nanoparticles in ultralow-volume blood samples with low concentrations, making it a powerful tool for analyzing and understanding the fate of nanoparticles in vivo.

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis.

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics.

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.

A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface.

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as ß-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

At-line N-linked glycan profiling for monoclonal antibodies with advanced sample preparation and high-performance liquid chromatography.

N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) system to achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs.

Recent advances in robotic protein sample preparation for clinical analysis and other biomedical applications.

Discovery of new protein biomarker candidates has become a major research goal in the areas of clinical chemistry, analytical chemistry, and biomedicine. These important species constitute the molecular target when it comes to diagnosis, prognosis, and further monitoring of disease. However, their analysis requires powerful, selective and high-throughput sample preparation and product (analyte) characterisation approaches. In general, manual sample processing is tedious, complex and time-consuming, especially when large numbers of samples have to be processed (e.g., in clinical studies). Automation via microtiter-plate platforms involving robotics has brought improvements in high-throughput performance while comparable or even better precisions and repeatability (intra-day, inter-day) were achieved. At the same time, waste production and exposure of laboratory personnel to hazards were reduced. In comprehensive protein analysis workflows (e.g., liquid chromatography-tandem mass spectrometry analysis), sample preparation is an unavoidable step. This review surveys the recent achievements in automation of bottom-up and top-down protein and/or proteomics approaches. Emphasis is put on high-end multi-well plate robotic platforms developed for clinical analysis and other biomedical applications. The literature from 2013 to date has been covered.

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in Xenopus laevis Embryos.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm2 surface area, reduces the total volume of reagents to <10 µL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 µg protein homogenates.

Evaluation and optimization of sample pretreatment for GC/MS-based metabolomics in embryonic zebrafish.

Metabolomics tactics have been applied in the research associated with embryonic zebrafish. However, the report regarding the evaluation of impacts of sample pretreatment on metabolomics results from zebrafish embryos is limited. In the present study, different data normalization approaches, extraction solvents, and extraction strategies for off-line derivatization gas chromatography coupled with mass spectrometry-based metabolomics analysis of zebrafish eleutheroembryos were evaluated and optimized. The results showed that, when 4-chlorophenylalanine normalization, sample homogenization and pure methanol combined with ultrasonic extraction were conducted, better repeatabilities, higher signals and broader coverages of detected metabolites can be achieved. The recovery and standard deviation of most standards were in the range of 82%-121% and 6.6%-12%, respectively, while the relative standard deviation of major detected metabolites ranged from 5.4% to 19%, indicating good extraction efficiencies and method precision. Under the developed method, 87 important endogenous metabolites such as citric acid and hypoxanthine were identified by universal databases or standards among 270 extracted metabolites, which consisted of sugars, amines, amino acids, nucleotides, fatty acids, and sterols. Therefore, the results could provide a proper pretreatment protocol for the analysis of wide-coverage metabolome in embryonic zebrafish. In addition, this study highlights the impact of normalization and extraction methods on the data quality of metabolomics analysis.

Impact of recovery correction or subjecting calibrators to sample preparation on measurement uncertainty: PAH determinations in waters.

The decision on the fitness of a measurement for its intended use and the interpretation of an analytical result requires the assessment of the measurement uncertainty. Frequently, the determination of analytes in complex matrices involves demanding sample preparations in which analyte losses are observed. These losses should be considered when reporting the results, which can be corrected for low recovery by taking the mean recovery observed in the analysis of reference items (e.g. spiked samples) or, alternatively, by subjecting calibrators to the same pre-treatment performed on the samples. In these cases, neat (NC) or adjusted (AC) calibrators are used, respectively. The way analyte losses are handled impacts on the measurement uncertainty. The top-down evaluation of the measurement uncertainty involves combining precision, trueness and additional uncertainty components. The trueness component is quantified by pooling various analyte recovery determinations. This work assesses and compares the uncertainty of polycyclic aromatic hydrocarbons (PAHs) measurements in water based on HPLC-FD calibrations with NC or AC. The trueness component is estimated by pooling mean recoveries observed from the analysis of different spiked samples to which mean recovery uncertainty and degrees of freedom are used to estimate a weighted mean recovery and respective uncertainty. The performance of measurements based on NC and AC are associated with equivalent uncertainty except when large analyte losses are observed, namely in the determination of Naphtalene. In this case, the processing of AC reduces the expanded relative uncertainty from 9.9% to 3.5%. The evaluated expanded uncertainty ranged from 3.5% to 12% of the measured value.