Optimized Sample Preparation Research Articles

Polycyclic aromatic hydrocarbons and petroleum products in soil samples of urban areas in Eastern Siberia

Introduction. In industrial cities, the problem of environmental pollution by supertoxicants, which include the class of polycyclic aromatic hydrocarbons (PAHs), is especially acute. Since among PAHs there are substances that have a carcinogenic effect, their quantitative identification in environmental objects is an urgent task.
 Materials and methods. The content of PAHs and petroleum products (PP) in the soil of an industrial city, taken in various functional zones, was studied. Determination of sixteen PAHs was carried out by gas chromatography-mass spectrometry (GC-MS) with optimized sample preparation. It is proposed to determine PAHs by GC-MS using dispersive liquid-liquid microextraction (DLLME). Petroleum products (PP) were determined by the fluorimetric method.
 Results. Total PAHs content (ΣPAH) varied from 7.50 to 319.11 µg/kg. ΣPAH-carcinogens in the residential and sanitary protection zones was on average 25.1–31.2 times higher than in the background site. PP content levels in urban soils ranged from 3.0 to 62.4 mg/kg.
 Limitations. The study is limited to the study of the upper layers of soil up to 5 cm. The possibility of PAH migration into groundwater has not been studied.
 Conclusion. The structure of PAHs was dominated by 4-6-nuclear structures: dibenz(a,h)anthracene, benzo(g,h,i)perylene, fluoranthene, benzo(b)fluoranthene, etc., making up > 87% of the total amount of PAHs. The proportion of these polyarenes in the total ΣPAH in the residential area increases by 1.3 times compared to the background, which indicates the possibility of their technogenic origin. The PAH and PP indicators are characterized by a scatter of data and, as a result, 
 a large influence of the local factor on the formation of soil cover pollution.

Comparative study of the ELISA use with milk and blood serum for bovine brucellosis diagnosis

Enzyme immunoassay, being a more sensitive method, makes it possible to identify specific antibodies in samples of combined milk. The ELISA study of harvested milk in farms can become an important element in the system of anti-brucellosis measures regarding the control of the epizootic state in farms taking into account the availability and ease of implementation of this method. For screening express-diagnostics of bovine brucellosis the method of enzyme immunoassay with milk of cows has been developed. ELISA with milk serum is specific, sensitive, easy to formulate, account for and interpret the results. It has been found that the conditions of storage and transportation of milk samples corresponding to room temperature values and leading to fermentation do not affect the level of specific anti-viral immunoglobulins for eight days, and the question of the use of a cold chain during transportation of milk samples to be examined to the place of analysis can be withdrawn from the agenda. When working out the optimal sample preparation, the difference in the specific signal when setting the ELISA between the milk serum obtained by high-speed centrifugation and the milk serum obtained by fermentation for 24 hours was less than 10%. Therefore, high-speed centrifugation was chosen for the preparation of milk serum samples during the ELISA study. The possibilities of using ELISA with milk serum on vaccinated and non-vaccinated cattle against brucellosis have been studied. Milk and blood for testing in the ELISA should be taken 6 months or more after vaccination (within the instructional time frame). A high level of correlation was established between ELISA data with milk and ELISA data with blood serum, regardless of epizootic or immune status (satisfactory and unfavorable herds, vaccinated and unvaccinated animals), which amounted to 86.8–92.0%.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

Strategies in sample preparation for proteome analysis of biological fluids from water buffaloes

Livestock species such as water buffalo have some unique characteristics like high fat percentage in milk and plasma, poor sample generation, peculiar behaviour, etc. making it difficult for proteomic analysis of such samples. Additionally, there is a limited database in terms of identified proteins, characterised antibodies and functional attributes of known molecules specific to buffalo. The body fluids like blood, milk, urine and foetal fluids offer a great opportunity in understanding physio-pathological processes and thus aid in developing reliable diagnostics. Standardised sample preparation protocols are key to advancement of proteomics science. Optimising sample preparation for 2-DE proteomics in buffalo serum, milk, and foetal fluids is crucial. Commercially available kits or laboratory methods for clean-up, enrichment, and condition optimising can be applied for getting optimum results. The 2-D clean up strategy is an important step in sample preparation procedures to get rid of inferring substances which hamper IEF. The sample preparation of the buffalo biological fluids (serum, foetal fluids, maternal uterine flushing and milk samples) reported in the present study shall provide valuable information for further research.

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Determination of persistent organochlorine pollutants in human serum by combining liquid-liquid extraction and solid-phase extraction with gas chromatography-tandem mass spectrometry

Persistent organochlorine pollutants (POCPs) are a group of “forever chemicals” that are ubiquitous in the environment and pose adverse health effects to humans. However, the lack of sensitive and widely applicable analytical method for internal POCPs exposure assessment puts limits on the researches of health risk evaluations. Therefore, we developed a method for the measurement of 43 POCPs in human serum using gas chromatography-tandem mass spectrometry (GC-MS/MS). Serum samples were treated with methanol and acid to precipitate protein and remove lipids, respectively. 13C-labeled analogue was spiked and liquid-liquid extraction (LLE) was performed to extract target analytes by hexane and methyl tert-butyl ether. The extract was purified by solid-phase extraction (SPE) and eluted with dichloromethane. The eluate was concentrated under nitrogen stream and reconstituted with toluene. Samples were analyzed by GC-MS/MS operated in electron impact (EI) and multiple reaction monitoring (MRM) mode. Calibration curves of all target analytes showed favorable linearity within the concentration range of 0.008 to 10 μg/L for different POCPs, with regression coefficients above 0.991. The method detection limits and limits of quantification ranged from 0.005 to 0.10 μg/L and 0.017 to 0.34 μg/L, respectively. The recoveries and precision of all analytes at 3 different spike levels were in the range of 65.3 % to 121.8 % and 1.8 % to 13.9 %, respectively. The matrix effects (ME) ranged from 74.9 % to 125.6 %. The results of measuring quality control samples from the National Institute of Standards and Technology (NIST) were with relative deviations of less than 30 %. With an optimized sample preparation procedure, the proposed method has successfully achieved in improving extraction efficiency and reducing ME. It was applied in the simultaneous determination of POCPs in a small volume of human serum in China National Human Biomonitoring.

Analysis of cerium-substituted yttrium iron garnets by inductively coupled plasma atomic emission spectrometry with preliminary microwave decomposition

An analytical procedure for inductively coupled plasma atomic emission (ICP-AES) analysis of iron garnets of the composition Y3 – xCexFe5 – yGayO12, where x = 0.4 – 0.5 and y = 2.4 – 2.6 with preliminary microwave decomposition of the sample is described. The compositions of acid mixtures (HCl/HNO3), as well as modes of microwave heating (holding time and temperature), providing complete dissolution of the samples are proposed. In addition to optimized sample preparation, conditions for ICP-AES analysis of cerium-doped yttrium iron garnets were studied. The effect of matrix components (Y, Ce, Fe, Ga) on the determination of doping elements was revealed and studied. The operating parameters of the spectrometer (ICP power and nebulizer flow rate) have been substantiated, which make it possible to reduce the matrix effect of Fe, Ga, Y, Ce on the elements Na, Mg, Al, Si, P, K, Ca, Sc, Cr, Mn, Co, Ni, Cu, Zn, Se, Cd, Sn, Te, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Pb in iron yttrium garnets by the ICP-AES. ICP-AES measurements were performed in the axial and radial mode for trace elements and in the radial mode for matrix elements. The limits of quantification (LOQs) of most elements ranged within n × 10–5 – n × 10–4 wt.%. The accuracy is confirmed by the spike recovery test. The relative standard deviation is in the range of 1 – 5% depending on the selected plasma view mode and the analyzed analytes. The developed method makes it possible to determine macro- and microcomponents in yttrium iron garnets in a wide range of concentrations with a high accuracy.

BIOM-46. STANDARDIZATION OF LIQUID BIOPSY FOR PEDIATRIC DIFFUSE MIDLINE GLIOMA USING DDPCR

Abstract BACKGROUND Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A >T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. METHODS DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A >T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). RESULTS We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. CONCLUSION ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Optimization of sample preparation and data evaluation techniques for X‐ray fluorescence prediction of soil texture, pH, and cation exchange capacity of loess soils

AbstractUse of X‐ray fluorescence (XRF) spectrometry for estimation of soil texture, pH, and cation exchange capacity (CEC) is desirable given the time‐consuming nature of traditional methods. Recent studies have shown promising results; however, further investigation is required to determine the effects of sample preparation and data evaluation techniques on accuracy. Our objective was to compare (I) a simple but well‐founded approach, combining measurement of powder samples and modeling with elemental contents as predictors in stepwise multiple linear regressions (MLR), with alternative approaches including (II) use of partial least squares regression (PLSR), (III) sample preparation as a pressed pellet, and (IV) spectral intensities as predictors (20 kV, 40 kV, and concatenated 20 + 40 kV). A total of 395 loess soils from three arable fields were used with a fivefold random training‐testing approach and a hold‐one‐site‐out training‐testing approach. With random partitioning, clay, silt, and sand accuracy with approach I was excellent (ratio of performance to interquartile distance in validation (RPIQv) = 8.5–12.9), while pH and CEC estimations were satisfactory to excellent (RPIQv = 2.0–2.5 and 2.2–3.3, respectively). Differences between MLR and PLSR were negligible. Increases in accuracy with pellet samples were 1%–13% of RPIQv for 20 kV intensities, but effects were inconsistent for other predictors. The optimal predictor varied by property, and differences ranged from 3% to 13% of RPIQv. Improvements to accuracy from Approach I to the best alternative were largest for texture (10%–15%) but may be superfluous given the excellent accuracy across all approaches. Although the leave‐one‐site‐out training resulted in variable performance, inclusion of soils from the target site in training assured reliable accuracy.

Detection limit of FT-IR-based bacterial typing based on optimized sample preparation and typing model

Accurate and efficient bacterial typing methods are crucial to microbiology. Fourier transform infrared (FT-IR) spectroscopy enables highly distinguishable fingerprint identification of closely related bacterial strains by producing highly specific fingerprints of bacteria, which is increasingly being considered as an alternative to genotypic methods, such as pulsed field gel electrophoresis (PFGE) and whole genome sequencing (WGS), for bacterial typing. Compared with genotypic methods, FT-IR has significant advantages of convenient operation, fast speed, and low cost. Fundamental research into the detection limit based on optimized analytical conditions for FT-IR bacterial typing, which can avoid excessive bacterial culture time or sampling volume, is particularly important, especially in clinical practice. However, the corresponding parameters have not been fully investigated. In this study, we developed a simplified and reliable procedure for sample preparation, optimized the data analysis procedure, and evaluated the FT-IR detection limit based on the above conditions. In particular, we combined the film mold and calcium fluoride plate for sample preparation. We evaluated the detection limit (about 108 CFU/mL) after parameter optimization using hierarchical cluster analysis (HCA) and artificial neural network (ANN). The optimization and evaluation of these key fundamentals will better promote future application of FT-IR-based bacterial typing.

Establishment of a quantitative method for GC analysis of polyoxyethylene (35) castor oil in microemulsion extracts

With the continuous exploration of microemulsions as solvents for traditional Chinese medicine extraction, polyoxyethy-lene(35) castor oil(CrEL), a commonly used surfactant, is being utilized by researchers. However, the problem of detecting residues of this surfactant in microemulsion extracts has greatly hampered the further development of microemulsion solvents. Based on the chemical structures of the components in CrEL and the content determination method of castor oil in the 2020 edition of the Chinese Pharmacopoeia(Vol. Ⅳ), this study employed gas chromatography(GC) and single-factor experiments to optimize the preparation method of methyl ricinoleate from CrEL. The conversion coefficient between the two was validated, and the optimal sample preparation method was used to process microemulsion extracts of Zexie Decoction from three batches. The content of methyl ricinoleate generated was determined, and the content of CrEL in the microemulsion extracts of Zexie Decoction was calculated using the above conversion coefficient. The results showed that the optimal preparation method for CrEL was determined. Specifically, 10 mL of 1 mol·L~(-1) KOH-methanol solution was heated at 60 ℃ for 15 min in a water bath. Subsequently, 10 mL of boron trifluoride etherate-methanol(1∶3) solution was heated at 60 ℃ for 15 min in a water bath, followed by extraction with n-hexane twice. CrEL could stably produce 20.84% methyl ricinoleate. According to this conversion coefficient, the average mass concentration of CrEL in the three batches of Zexie Decoction microemulsion extracts was 11.94 mg·mL~(-1), which was not significantly different from the CrEL mass concentration of 11.57 mg·mL~(-1) during microemulsion formulation, indicating that the established content determination method of this study was highly accurate, sensitive, and repeatable. It can be used for subsequent research on microemulsion extracts of Zexie Decoction and provide a reference for quality control of other drug formulations containing CrEL.

Fast Forward: Optimized Sample Preparation and Fluorescent Staining for Microplastic Detection

The fast, affordable, and standardized detection of microplastics (MP) remains one of the biggest challenges in MP research. Comparable data are essential for appropriate risk assessments and the implementation of laws and limit values. The fluorescent staining of MP in environmental samples is a possible solution to this problem. This study investigates the optimization of a sample preparation process (hydrogen peroxide digestion) and the staining process (temperature, concentration, time, surfactants as staining aids) for using a specifically developed fluorescent dye for MP detection. The optimization is performed by comparing the sample preparation process and staining of MP from different polymers and natural particles. Further, the suitability of the optimized process for the detection of fluoropolymers and tire abrasion was tested. The results show that the optimized method (increased temperature and optimized stain concentration) can detect microplastics reliably with a total sample preparation and measurement time of 2.5–3 h per sample, reaching recovery rates of 93.3% (polypropylene) to 101.7% (polyester). Moreover, two of the three tested fluoropolymers could be detected reliably. Tire abrasion could not be detected with the here presented method, as the black color leads to strong quenching. A long-term study measuring the MP pollution in the effluent of a municipal wastewater treatment plant compared the optimized and original processes and confirmed the stability of the improved method for routine measurements and contamination control.

Sample preparation and analysis protocols for the elucidation of structure and chemical distribution in kidney stones

Examining intact kidney stones both qualitatively and quantitatively can be difficult due to their size and fragility. Many modern analysis methods often lead to the destruction of the stone's structure during sample preparation. Preserving the structural integrity is crucial for accurately determining the chemical distribution of the components of kidney stones, which, in turn, improves our understanding of the disease's etiology. Infrared microspectroscopy and imaging play a vital role in revealing the stone's microstructure and component distribution. Consequently, this research focuses on investigating the impact of different sample preparation techniques on kidney stone analysis using infrared microspectroscopy. Specifically, it explores how polishing the surface of cross-sectioned stones influences the results. The polishing was performed utilizing abrasive discs and lapping films. A polishing device was also designed for the optimization of sample preparation. Additionally, this work involved a comparison of reflection infrared imaging with Attenuated Total Internal Reflection (ATR) infrared microspectroscopic imaging for the analysis of the microstructure of urinary stones.

Erratum: Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae.

An erratum was issued for:Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae. The Authors section was updated from: Gan Guangming1,2 Sheng Qingyuan3 Ou Yutong1 Chen Mei1 Zhang Chenchen1 1The School of Medicine, Southeast University, 2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 3The School of Life Science and Technology, Southeast University Corresponding Authors:Gan Guangming to: Gan Guangming1,2 Sheng Qingyuan3 Ou Yutong1 Chen Mei3 Zhang Chenchen1 1The School of Medicine, Southeast University, 2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 3The School of Life Science and Technology, Southeast University Corresponding Authors:Gan Guangming,Sheng Qingyuan.

Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy.

In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.

Selectivity of β-Cyclodextrin Polymer toward Aquatic Contaminants: Insights from Ultrahigh-Resolution Mass Spectrometry of Dissolved Organic Matter.

Selectivity in solid-phase extraction (SPE) materials has become increasingly important for analyte enrichment in sensitive analytical workflows to alleviate detrimental matrix effects. Molecular-level investigation of matrix constituents, which are preferentially extracted or excluded, can provide the analytical chemist with valuable information to learn about their control on sorbent selectivity. In this work, we employ nontargeted Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to elucidate the molecular chemodiversity of freshwater-derived dissolved organic matter (DOM) extracted by the selective model sorbent β-cyclodextrin polymer (β-CDP) in comparison to conventional, universal SPE sorbents (i.e., Oasis HLB, Supel-Select HLB, and LiChrolut EN). Statistical analysis of MS data corroborated the highly selective nature of β-CDP by revealing the extracted DOM spectra that are most dissimilar to original compositions. We found that its selectivity was characterized by pronounced discrimination against highly oxygenated and unsaturated DOM compounds, which were associated with the classes of lignin-like, tannin-like, and carboxylic-rich alicyclic molecules. In contrast, conventional sorbents excluded less highly oxygenated compounds and showed a more universal extraction behavior for a wide range of DOM compositional space. We lay these findings in a larger context that aids the analyst in obtaining an a priori estimate of sorbent selectivity toward any target analyte of interest serving thereby an optimization of sample preparation. This study highlights the great value of nontargeted ultrahigh-resolution MS for better understanding of targeted analytics and provides new insights into the selective sorption behavior of novel sorbents.

First purported images of ciguatoxin-3C by cryogenic and transmission electron microscopy

Marine biotoxins in seafood products are responsible for human illnesses and seafood production losses. Ciguatera poisoning (CP) is the most commonly reported non-bacterial seafood-related illness worldwide and is caused by the consumption of fish or shellfish containing ciguatoxins (CTXs), a class of polycyclic polyether marine biotoxins with highly complex chemical structures. Reliable laboratory testing in clinical samples for CTXs is not available and many analogues remain structurally unresolved. CTXs have a proposed backbone size of 3 nm but have never been directly observed. Herein, the first purported images of CTX-3C obtained by Cryogenic Electron Microscopy (Cryo-EM) and in the presence of gold nanoparticles with attached antibodies by Transmission Electron Microscopy (TEM) are presented. TEM image was captured based on a sample preparation optimization technique using anti-ciguatoxins CTX-3C [1C49] Ab00350–1.1 purified antibodies, gold nanoparticles, and a concentration of synthetic CTX-3C molecules combined for imaging. We discuss how other existing instrumentations with atomic resolutions exceeding <0.1 nm could be applied using this preparatory method to observe, with greater detail, the common backbone of all known CTXs and its proposed wider adoption towards other marine biotoxins. Conventional detection methods for marine toxins have drawbacks, applying cutting-edge existing technologies such as TEMs to directly observe never-before-seen marine toxins can contribute to the fundamental science of marine toxins research.

Rapid and in-depth proteomic profiling of small extracellular vesicles for ultralow samples.

The integration of robust single-pot, solid-phase-enhanced sample preparation with powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used to define the extracellular vesicle (EV) proteome landscape and underlying biology. However, EV proteome studies are often limited by sample availability, requiring upscaling cell cultures or larger volumes of biofluids to generate sufficient materials. Here, we have refined data independent acquisition (DIA)-based MS analysis of EV proteome by optimizing both protein enzymatic digestion and chromatography gradient length (ranging from 15 to 44min). Our short 15min gradient length can reproducibly quantify 1168 (from as little as 500 pg of EV peptides) to 3882 proteins groups (from 50ng peptides), including robust quantification of 22 core EV marker proteins. Compared to data-dependent acquisition, DIA achieved significantly greater EV proteome coverage and quantification of low abundant protein species. Moreover, we have achieved optimal magnetic bead-based sample preparation tailored to low quantities of EVs (0.5 to 1µg protein) to obtain sufficient peptides for MS quantification of 1908-2340 protein groups. We demonstrate the power and robustness of our pipeline in obtaining sufficient EV proteomes granularity of different cell sources to ascertain known EV biology. This underscores the capacity of our optimised workflow to capture precise and comprehensive proteome of EVs, especially from ultra-low sample quantities (sub-nanogram), an important challenge in the field where obtaining in-depth proteome information is essential.

Analysis of PCDD/Fs in environmental samples by using gas chromatography in combination with high resolution mass spectrometry: optimization of sample preparation

ABSTRACT This study developed a simple, economical and time-saving clean-up procedure based on an in-house packed column system for the determination of the most toxic-relevant 17 PCDD (polychlorinated dibenzo-p-dioxin) and PCDF (polychlorinated dibenzofuran) congeners in environmental samples following accelerated solvent extraction. The extract solution was cleaned up using a manually packed column system consisting of a multi-layer silica gel column and an activated carbon column. After clean-up, dioxins and furans were analysed by using gas chromatography in combination with high-resolution mass spectrometry. Isotopic labelled spiking experiments investigated the recovery of all dioxin and furan congeners. Then, the clean-up procedure for environmental samples was validated by applying the analysis of both clean soil reference material (CRM EDF-5183) and heavily contaminated sediment reference material (CRM EDF-5184) samples, as well as participating in the proficiency test (PT) for sediment samples. The recovery efficiency of isotope-labelled standard compounds in certified reference material (CRM) samples and PT samples ranged from 35% to 101% and between 46.1% and 105%, respectively. The mass concentrations of 17 PCDD/Fs in CRM samples were in the acceptable range. The absolute z-score value for each PCDD/F congener in the PT samples was below 1, confirming the analytical procedure’s high accuracy. The applied clean-up procedure in this study has demonstrated accuracy, repeatability and a significant increase in sample extraction/preparation productivity by reducing the time for sample preparation.

B-290 Development of a LCMS/MS Method for the Quantitation of Infliximab in Serum

Abstract Background Quantitative bottom-up proteomics of biotherapeutics has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. The general workflow includes multiple steps: sample extraction/purification, reduction, alkylation, enzymatic digestion, desalting, and LC-MS/MS analysis of signature peptides. In this study, we developed an infliximab drug assay with desired operational efficiencies needed in a clinical laboratory. Evaluations included reduction/alkylation removal, trypsin digestion length, trypsin quality, online/offline/no sample desalting, multiple/single signature peptides, and whole protein/peptide internal standardization. Methods For sample preparation, a general procedure was initially followed. Briefly, protein precipitation with ammonium sulfate, re-suspension with buffer, reduction, alkylation, overnight enzymatic digestion, desalting, and LC-MS/MS analysis. To simplify the workflow, the steps of reduction and/or alkylation were omitted. The trypsin digestion time was varied from 2 h, 4 h, and overnight. TPCK-modified trypsin from two different vendors (Thermo Scientific and Millipore Sigma) were also compared for digestion efficiency. SILu™MAb infliximab stable-isotope labeled monoclonal antibody (Millipore sigma) was compared with customer synthesized stable-isotope labeled signature peptides as the internal standard. Different trapping cartridges were evaluated for online sample desalting and different analytical columns were evaluated for target peptide analysis. A Transcend TurboFlow TLX UHPLC System coupled to a TSQ Altis triple-quadrupole mass spectrometer was used (Thermo Scientific) operating in multiple reaction monitoring mode (2 transitions for each peptide and internal standard). Two signature peptides YASE (Sequence: YASESMSGIPSR) and GLEW (Sequence: GLEWVAEIR) from different chains of infliximab were analyzed. Results For sample preparation, the omission of protein reduction/alkylation from the workflow did not significantly affect YASE quantitation. The peak area of GLEW decreased by about 30% without reduction/alkylation. The evaluation of three calibration curves corresponding to the digestion time of 2, 4, and overnight respectively showed that 4 h was optimal for all the levels of calibrators if both signature peptides were used for quantitation. TPCK-modified trypsin from two different vendors were equal in digestion efficiency. The calibration curve using SILu™MAb infliximab stable-isotope labeled monoclonal showed superior linearity (R2 &amp;gt; 0.98) and recovery for each calibrator vs using stable-isotope labeled signature peptides as internal standard. With the optimized conditions, both signature peptides can be used for the accurate quantitation of infliximab. However, for monitoring both peptides simultaneously, the chromatography separation needed 14.5 min. On the contrary, 9 min is needed for monitoring a single peptide YASE. Conclusion In summary, systematic optimization of sample preparation and LC-MS/MS analysis was accomplished in this study. The entire workflow was simplified by removing the steps of reduction and alkylation and using optimal digestion time, which increases the efficiency and significantly facilitate the implementation of this assay.