High Sample Throughput Research Articles

Single Cell Phenotypic Analysis for Cancer Stem Cell Identification by Dual-Isotope ICP-QMS.

Single cell phenotypic analysis is significant for clinical diagnosis, treatment, and prognosis of cancer. Accurate differentiation of cancer stem cell (CSC) subpopulations from a large number of cancer cells may become a cancer surveillance tool and provide important implications for the development of new CSC-targeted therapy strategies. Herein, we report a new approach based on dual-isotope inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) for single cell phenotypic analysis. High-throughput single cell sampling was achieved by a spiral channel microfluidic chip for cell focusing and alignment, and single cell analysis was performed with time-resolved ICP-QMS by identifying the highly specific probes. This enables the monitoring of two surface protein markers (EpCAM and MUC1) of three cell types, i.e., HeLa, MCF-7, and HepG2, at single cell level. The analysis of breast cancer stem cells further confirmed its capability in distinguishing rare cell phenotypes. The present study provides promising possibilities for adopting ICP-QMS in biomedical investigations in terms of cell typing, stemness identification of tumor cells, and cell heterogeneity analysis.

Innovative micellar-UPLC approach for concomitant determination of favipiravir and remdesivir with four co-administered cardiovascular drugs in their pharmaceuticals and spiked plasma: Greenness and whiteness assessment

Many COVID-19 patients have pre-existing cardiovascular diseases, which are strongly linked to the high incidence of severe COVID-19. The experts advised such patients to prioritize drug treatments as per the physician's recommendations to avoid aggravation of the basic diseases. Hence, the development of an analytical approach for the concurrent determination of drugs prescribed for the co-treatment of COVID-19 and cardiovascular diseases acquires significant importance in QC units and bioavailability centers. Herein, a micellar-UPLC approach was first devised for the concomitant analysis of aspirin, atenolol, atorvastatin, losartan, favipiravir, and remdesivir as co-administered drugs, either with or without salicylic acid. On ACQUITY UPLC C18 column, the aforesaid drugs were isocratically separated employing a micellar eluent system. This system consisted of 0.03 M Brij-35, 0.15 M sodium dodecyl sulfate, and 0.02 M sodium dihydrogen phosphate (pH 5.0) as the mobile phase A (90%) and n-propanol as the mobile phase B (10%). The eluent system was pumped at a flow rate of 0.50 mL.min−1 for 5.50 min, which was suddenly increased to 0.80 mL.min−1 for 2.50 min. The separated peaks were scanned at 240 nm. The suggested approach was validated following ICH requirements for linearity, robustness, accuracy, specificity, precision, and detection and quantitation limits. The suggested approach was applied successfully to commercial products and human plasma without any intrusion from excipients or plasma matrices. The suggested approach’s greenness was appraised employing five metrics namely: eco-scale, GAPI, NEMI, Raynie and Driver, and AGREE. Furthermore, the suggested approach’s whiteness features were investigated using the recently unveiled RGB12 model. The suggested approach outperformed the reported approaches in terms of greenness and whiteness. The sensitivity, high sample throughput, and short runtime (7.40 min) of the suggested approach assert its aptness for routine determination of the aforesaid drugs.

Direct Determination of the Aluminum Content in Antiperspirants Using Digital Image Colorimetry

AbstractIn this work a dilute‐and‐shoot procedure using digital image colorimetry (DIC) was evaluated for the determination of aluminum in antiperspirants. Derivatization was performed by means of the reaction between Al(III) and Alizarin Red S (ARS) reagent. Digital images were acquired using a table scanner and the average values for red, green and blue channels (RGB) were obtained. Optimal conditions were set at pH=4.5, ARS concentrations 10‐fold higher than Al(III) and using the G channel. The method presented linearity in the 2.0–10.0 mg L−1 range, the limit of detection (LOD) was 1.1 mg L−1 and precision was better than 2.55 %. Accuracy was checked by the analysis of spiked samples at 2.0 and 4.0 mg L−1 Al(III), and good recoveries (82.6–117.5 %) were obtained. Eight samples of roll‐on antiperspirants were analyzed by the proposed colorimetric method and the well‐stablished flame atomic absorption spectrometry (FAAS) comparative method. The obtained results were not statistically different at 95 % confidence level. An analytical greenness assessment was performed for both methods and the procedure based on digital image colorimetry was proven to be a greener approach due to miniaturization, low sample consumption, least amount of waste and high sample throughput.

Automated Sample Preparation Workflow for Tandem Mass Tag-Based Proteomics.

Although tandem mass tag (TMT)-based isobaric labeling has become a powerful approach for multiplexed protein quantitation, automating the workflow for this technique has not been easy to achieve for widespread adoption. This is because preparation of TMT-labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction, and alkylation to tryptic digestion, desalting, labeling, and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic, especially with large-scale clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for an automated proteomic workflow employing TMT labeling with the manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using an automated platform and a conventional manual protocol. Single-shot liquid chromatography with tandem mass spectrometry analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiency of tryptic digestion, alkylation, and TMT labeling were similar in both manual and automated processes. In addition, comparison of quantitation accuracy and precision showed similar performance in an automated workflow compared to manual sample preparation by an expert. Overall, we demonstrated that the automated platform performs at a level similar to a manual process performed by an expert for TMT-based proteomics. We anticipate that this automated workflow will increasingly replace manual pipelines and has the potential to be applied to large-scale TMT-based studies, providing robust results and high sample throughput.

Development of sugars quantification method using polarimeter couple with UV-Visible quartz cuvette cell

The objective of this study was to develop the method for determination of sugars at low sample volume using UV-Visible quartz cuvette cuvett instead of polarimeter cell. This proposed method could be analysis of sugars with high sample throughput rate and appropriate for analysis of pharmaceutical formulation. This study, the simple determination method by using sample UV-Visible quartz cuvette was developed in order to analyze dextrose, sucrose and lactose. It was found that quartz cuvette was not affected from the sodium light source at wavelength of 589.0 nm and it was possible to adapt for polarimetry technique. The linear calibration curve of dextrose, sucrose and lactose standard solution using polarimeter sample tube were constructed in the concentration ranges of 2.5–15.0 % w/v. Linear regression between the angle of rotation (y) and concentration of each sample (x) were expressed as the equation of y = 1.0941x – 0.1824 (r2= 0.9993), y = 1.3515x –0.1581 (r2= 0.9998) and y= 1.0390x – 0.0162 (r2= 0.9997) for dextrose, sucrose and lactose, respectively. Interestingly, the calibration curve of sugars standard solution using UV-Visible quartz cuvette were shown as y = 0.1050x - 0.1038 (r2= 0.9990), y = 0.1312x – 0.1458 (r2= 0.9989) and y = 0.1072x – 0.0335 (r2= 0.9991) for dextrose, sucrose and lactose, respectively. The detection limits (LOD) of proposed method for determination of dextrose, sucrose and lactose were 0.5, 1.0 and 0.6 % w/v, respectively, while the quantitation limits (LOQ) were 1.6, 3.0 and 1.9 %w/v for anlysis of dextrose, sucrose and lactose, respectively. The interferences study for proposed method from sodium chloride was investigated. It had found that this salt shown none effect for the proposed method. The results acquired the proposed method were compared favorably with those acquired by the reference method at a 95% confidence level with no significant difference (n=10). The developed method had shown accurate, precise, and reproducible. Moreover, this method indicated the possibility of a modified polarimetric method for the quantification of sugars. The method is possible to apply both in simple quality control laboratory and local industrial that involved with pharmaceutical production.

Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates.

ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a 'goodness of fit' of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value.

Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples

ObjectiveGrowth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34–69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods.ResultsDiversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.

Development of a robust UPLC-MS/MS method for the quantification of riluzole in human plasma and its application in pharmacokinetics.

Introduction: The aim of the present study was to establish a simple method for the determination of riluzole in human plasma by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and apply it for the determination of riluzole in amyotrophic lateral sclerosis (ALS) patients. Methods: Samples were prepared by protein precipitation and were then gradient-eluted on a column of ACQUITY UPLC® HSS T3 by using 0.1% formic acid acetonitrile and 0.1% formic acid water as the mobile phase. Detection was performed on a Xevo TQ-S tandem mass spectrometer in the multiple-reaction monitoring mode using positive electrospray ionization. Validation was performed in the range of 5-800ng/mL. Results and discussion: Three batches of precision accuracy, selectivity, matrix effects, extraction recovery, and stability were also verified and met the requirements. The results showed that the method was reliable and successfully applied to the pharmacokinetics study of riluzole in Chinese amyotrophic lateral sclerosis patients. Meanwhile, in comparison with other prior published methods, our method has the advantages of simple sample preparation, relatively short running time, and small plasma sample consumption, which represented a high-throughput sample determination potential.

Development of a novel strategy for the quantification of ultra-trace impurity elements in high-purity magnesium using inductively coupled plasma tandem mass spectrometry

High purity magnesium is not only an important basic raw material for semiconductor and electronics industries, but also a promising new generation of electrochemical energy storage materials and biomedical materials. Impurities in high-purity magnesium affect material properties, which has become the most critical factor restricting its application. However, accurate analysis of multiple ultra-trace impurity elements in high-purity magnesium is extremely challenging. In this paper, based on the synergistic effect of N2O/H2 reaction gas mixture to eliminate spectral interference of inductively coupled plasma tandem mass spectrometry (ICP-MS/MS), a new strategy for the quantification of 45 ultra-trace impurity elements in high-purity magnesium was proposed. The results indicated that the limits of detection (LOD) were in the range of 0.02–18.5 ng L − 1; the LODs of the challenging non-metallic elements Si and S were 18.5 and 12.2 ng L − 1, respectively; and the LODs of all the other analytes were less than 10 ng L − 1. Even under hot plasma conditions, LODs of alkali metal elements were also less than 5 ng L − 1. The spike recovery of each analyte was 93.6%–107%, and the relative standard deviation (RSD) was 3.2%–6.9%, respectively. At a 95% level of confidence, no significant differences were found between the results obtained under the optimal conditions for the analyte with the developed method and the measurement results of SF-ICP-MS. The developed method indicated low LOD, high sample throughput, and complete interference elimination, demonstrating a new avenue for the rapid determination of ultra-trace elements in high-purity magnesium.

Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress.

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6-7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n=3) and sample (n=100) n-plex capacities in ∼4h with 50-70min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

A high-throughput pipeline for DNA/RNA/small RNA purification from tissue samples for sequencing.

High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNAand small RNA, calledGSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recoveryis presented. GRAB-ALL wasbenchmarkedagainst existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.

A high-throughput mass cytometry barcoding platform recapitulating the immune features for HCC detection

Early diagnosis of hepatocellular carcinoma (HCC), a highly malignant disease, is crucial for improving patient prognosis. Liquid biopsy has emerged as a promising approach for cancer detection, especially peripheral immunity profiling using mass cytometry time-of-flight (CyTOF). While the limitied throughput and high cost hamper its widespread application. To address these challenges, we developed an automated micro-droplet printing platform to achieve high-throughput mass cytometry barcoding (MCB) strategy. Utilizing a poly (methyl methacrylate) (PMMA) plane substrate, we established a multiplex barcoding checkboard in binary labeling strategy through “plate covering”. To validate the utility of our system, we applied the multiplex barcoding system to label human peripheral blood samples from two cohorts consisting of 16 and 64 samples respectively, while successfully recapitulating the immune features associated with HCC. The ability of high-throughput barcoding samples empowers the application of CyTOF for large-scale HCC detection and other potential uses.

Evaluating cPILOT Data toward Quality Control Implementation.

Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.

EwaldSolidSolution: A High-Throughput Application to Quickly Sample Stable Site Arrangements for Ionic Solid Solutions.

Data-driven materials design of ionic solid solutions often requires sampling (meta)stable site arrangements among the massive number of possibilities, which has been hampered by the lack of relevant methods. Herein, we develop a quick high-throughput sampling application for site arrangements of ionic solid solutions. Given the Ewald Coulombic energies for an initial site arrangement, EwaldSolidSolution updates the modified parts of the energy with varying sites only, which can be exhaustively estimated by using massively parallel processing. Given two representative examples of solid electrolytes, Li10GeP2S12 and Na3Zr2Si2PO12, EwaldSolidSolution successfully calculates the Ewald Coulombic energies of 211,266,225 (235,702,467) site arrangements for Li10GeP2S12 (Na3Zr2Si2PO12) with 216 (160) ion sites per unit cell in 1223.2 (1187.9) seconds: 0.0057898 (0.0050397) milliseconds per site arrangement. The computational cost is enormously saved in comparison with an existing application, which estimates the energy of a site arrangement on the second timescale. The positive correlations between the Ewald Coulombic energies and those estimated by density functional theory calculations show that (meta)stable samples are easily revealed by our computationally inexpensive algorithm. We also reveal that the different-valence nearest-neighbor pairs are distinctively formed in the low-energy site arrangements. EwaldSolidSolution will boost the materials design of ionic solid solutions by attracting broad interest.

Binding-and-Folding Recognition of an Intrinsically Disordered Protein Using Online Learning Molecular Dynamics.

Intrinsically disordered proteins participate in many biological processes by folding upon binding to other proteins. However, coupled folding and binding processes are not well understood from an atomistic point of view. One of the main questions is whether folding occurs prior to or after binding. Here we use a novel, unbiased, high-throughput adaptive sampling approach to reconstruct the binding and folding between the disordered transactivation domain of c-Myb and the KIX domain of the CREB-binding protein. The reconstructed long-term dynamical process highlights the binding of a short stretch of amino acids on c-Myb as a folded α-helix. Leucine residues, especially Leu298-Leu302, establish initial native contacts that prime the binding and folding of the rest of the peptide, with a mixture of conformational selection on the N-terminal region with an induced fit of the C-terminal.

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study.

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49-246 ng/mg for caffeine abusers, and 0.25-363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection.

An Atomic Force Microscope(AFM) isa powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers ("Quattro") with a 125 µm tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

A novel method of high-throughput micro-and nanomechanical sample fabrication.

There is an increasing interest in understanding materials' mechanical properties at small length scales. Mechanical testing from nano- to meso-scale has seen a rapid development over the last decade, leading to a high demand of sample fabrication. In the present work, a novel method of micro-and nanomechanical sample preparation is introduced based on a new technique combining femtosecond laser and focused ion beam (FIB), namely LaserFIB. The new method greatly simplifies the sample preparation workflow by taking advantage of the fast milling-rate of femtosecond laser and the high precision of FIB. It significantly improves the processing efficiency and success rate, allowing for the high-throughput preparation of reproducible micro- and nanomechanical specimens. The novel method has far more advantages: (1) it allows for site-specific sample preparation based on scanning electron microscope (SEM) characterization (lateral and depth direction of bulk material) (2) following the new workflow, mechanical specimens are still connected to the bulk by its natural bonding, yielding more reliable mechanical testing results; (3) it extends the processable sample size to meso-scale while still remaining high precision and high efficiency; (4) the seamless transfer between laser and FIB/SEM chamber greatly reduces the risk of sample damage and is very friendly for environmental sensitive materials. The new method solves critical problems for high-throughput multiscale mechanical sample preparation, greatly contributing to the development of nano to meso-scale mechanical testing by making sample preparation efficient and convenient.