High-throughput Quantitative Analysis Research Articles

Quantitative analysis of the impurities in Etimicin using hydrophilic interaction liquid chromatography coupled with charged aerosol detector

Etimicin is a typical aminoglycoside antibiotic (AG). High performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD) method is a commonly used method for determining impurities in Etimicin. However, due to the poor reproducibility, low sensitivity and narrow linear range of the ELSD, high-throughput quantitative analysis of impurities in Etimicin currently poses a challenge. In this study, a sensitive method using hydrophilic interaction liquid chromatography coupled with charged aerosol detector (HILIC-CAD) was developed for the analysis of the impurities in Etimicin. The liquid phase conditions for determination impurities in Etimicin were optimized using Box Behnken design (BBD) and response surface methodology (RSM), resulting in satisfactory separation and optimal CAD output signal. We also studied the influence of CAD parameters on the signal-to-noise ratio and linearity of Etimicin and its impurities. This method has also been proven to be effective in separating impurities from two other typical AGs, Isepamicin and Amikacin. In the method validation, the coefficient of determination (R2) of Etimicin, Isepamicin and Amikacin and their impurities were all greater than 0.999, within the range of 0.5–50 μg/mL. The average recoveries of the impurities of three typical AGs were 99.03 %-101.22 %, RSDs all were less than 2.5 % for intra-day and inter-day precision, with good precision and accuracy. The developed HILIC-CAD quantification method was sensitive, accurate and highly selective for quantitative analysis of impurities in the AGs without need ion-pairing reagents, which is ensure the public medication safety. The method is first reported application of HILIC-CAD method for quantitative analysis of the impurities in AGs.

A Portable Automated Microfluidic Platform for Point-of-Care Testing for Multiple Mycotoxins in Wine.

Food safety requires point-of-care testing (POCT) for mycotoxins, since their presence in wine significantly impacts the wine industry and poses a severe threat to human life. Traditional detection methods are usually limited to detecting one mycotoxin and cannot achieve high-throughput, automated, and rapid quantitative analysis of multiple mycotoxins in real samples. Here, we propose a portable automated microfluidic platform (PAMP) integrating a chemiluminescence (CL) imaging system and a microfluidic chip to realize POCT for multiple mycotoxins in real samples, simplifying complex manual operations, shortening the detection time, and improving the detection sensitivity. Specially, silicone films were used as substrates on microfluidic chips to incubate mycotoxin conjugations, and the streptavidin-biotin (SA-B) system and an indirect immunoassay were implemented on silicone films to improve the sensitivity of reaction results. Interestingly, these methods significantly improved detection results, resulting in sensitive detection of mycotoxins, including zearalenone (ZEA) ranging from 1 to 32 ng/mL, aflatoxin B1 (AFB1) ranging from 0.2 to 6.4 ng/mL, and ochratoxin A (OTA) ranging from 2 to 64 ng/mL. The recovery of samples reached 91.39-109.14%, which verified the reliability and practicability of the PAMP. This PAMP enables sensitive and rapid detection of multiple mycotoxins in markets or wineries that lack advanced laboratory facilities. Therefore, it is essential to develop a portable microfluidic platform for POCT to detect mycotoxins in real samples.

Quantification of binding capacity of natural products to target proteins by sensors integrating SERS labeling and photocrosslinked molecular probes

Natural products-based screening of active ingredients and their interactions with target proteins is an important ways to discover new drugs. Assessing the binding capacity of target proteins, particularly when multiple components are involved, presents a significant challenge for sensors. As far as we know, there is currently no sensor that can accomplish high-throughput quantitative analysis of natural product-target protein binding capacity based on Raman spectroscopy. In this study, a novel sensor model has been developed for the quantitative analysis of binding capacity based on Surface-Enhanced Raman Spectroscopy (SERS) and Photocrosslinked Molecular Probe (PCMP) technology. This sensor, named SERS-PCMP, leverages the high throughput of molecular probe technology to investigate the active ingredients in natural products, along with the application of SERS labelling technology for target proteins. Thus it significantly improves the efficiency and accuracy of target protein identification. Based on the novel strategy, quantitative analysis of the binding capacity of 20 components from Shenqi Jiangtang Granules (SJG) to α-Glucosidase were completed. Ultimately, the binding capacity of these active ingredients was ranked based on the detected Raman Intensity. The compounds with higher binding capacity were Astragaloside IV (Intensity, 138.17), Ginsenoside Rh2 (Intensity, 87.46), Ginsenoside Rg3 (Intensity, 73.92) and Ginsenoside Rh1 (Intensity, 64.37), which all exceeded the binding capacity of the positive drug Acarbose (Intensity, 28.75). Furthermore, this strategy also performed a high detection sensitivity. The limit of detection for the enzyme using 0.1 mg of molecular probe magnetic nanoparticles (MP MNPs) was determined to be no less than 0.375 μg/mL. SERS-PCMP sensor integrating SERS labeling and photocrosslinked molecular probes which offers a fresh perspective for future drug discovery studies. Such as high-throughput drug screening and the exploration of small molecule-target protein interactions in vitro.

Characterization of lithium phosphorus oxide thin film libraries by laser-induced breakdown spectroscopy imaging: A step towards high-throughput quantitative analyses

Elemental analysis is a challenge for the development of High Throughput Experimentation (HTE) on thin film materials, and an even greater one when it comes to screening lithium-containing battery materials. In this regard, Laser-Induced Breakdown Spectroscopy has been evaluated here for the quantitative analysis of lithium in libraries of Li2.3PO3.65 amorphous solid electrolyte films. The LIBS analysis of multiple samples with the same composition, but with thicknesses ranging from 50 to 700 nm, has revealed a linear trend in the intensity ratio of the Li I 610.35 nm and P I 214.91 nm emission lines, opening the way to rapid quantitative analysis of material libraries. The sensitivity of the technique finally allowed the detection of the Li I 670.79 nm emission line for film thicknesses down to 4 nm, corresponding to 0.2% of the ablated volume, or about 0.15 pg of Li.

A Dried Blood Spot protocol for high-throughput quantitative analysis of SARS-CoV-2 RBD serology based on the Roche Elecsys system.

SARS-CoV-2 spreads pandemically since 2020; in 2021, effective vaccinations became available and vaccination campaigns commenced. Still, it is hard to track the spread of the infection or to assess vaccination success in the broader population. Measuring specific anti-SARS-CoV-2 antibodies is the most effective tool to track the spread of the infection or successful vaccinations. The need for venous-blood sampling however poses a significant barrier for large studies. Dried-blood-spots on filter-cards (DBS) have been used for SARS-CoV-2 serology in our laboratory, but so far not to follow quantitative SARS-CoV-2 anti-spike reactivity in a longitudinal cohort. We developed a semi-automated protocol or quantitative SARS-CoV-2 anti-spike serology from self-sampled DBS, validating it in a cohort of matched DBS and venous-blood samples (n = 825). We investigated chromatographic effects, reproducibility, and carry-over effects and calculated a positivity threshold as well as a conversion formula to determine the quantitative binding units in the DBS with confidence intervals. Sensitivity and specificity reached 96.63% and 97.81%, respectively, compared to the same test performed in paired venous samples. Between a signal of 0.018 and 250 U/mL, we calculated a correction formula. Measuring longitudinal samples during vaccinations, we demonstrated relative changes in titers over time in several individuals and in a longitudinal cohort over four follow-ups. DBS sampling has proven itself for anti-nucleocapsid serosurveys in our laboratory. Similarly, anti-spike high-throughput DBS serology is feasible as a complementary assay. Quantitative measurements are accurate enough to follow titer dynamics in populations also after vaccination campaigns. This work was supported by the Bavarian State Ministry of Science and the Arts; LMU University Hospital, LMU Munich; Helmholtz Center Munich; University of Bonn; University of Bielefeld; German Ministry for Education and Research (proj. nr.: 01KI20271 and others) and the Medical Biodefense Research Program of the Bundeswehr Medical Service. Roche Diagnostics provided kits and machines for analyses at discounted rates. The project is funded also by the European-wide Consortium ORCHESTRA. The ORCHESTRA project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101016167. The views expressed in this publication are the sole responsibility of the author, and the Commission is not responsible for any use that may be made of the information it contains.IMPORTANCESARS-CoV-2 has been spreading globally as a pandemic since 2020. To determine the prevalence of SARS-CoV-2 antibodies among populations, the most effective public health tool is measuring specific anti-SARS-CoV-2 antibodies induced by infection or vaccination. However, conducting large-scale studies that involve venous-blood sampling is challenging due to the associated feasibility and cost issues. A more cost-efficient and less invasive method for SARS-CoV-2 serological testing is using Dried-Blood-Spots on filter cards (DBS). In this paper, we have developed a semi-automated protocol for quantifying SARS-CoV-2 anti-spike antibodies from self-collected DBS. Our laboratory has previously successfully used DBS sampling for anti-nucleocapsid antibody surveys. Likewise, conducting high-throughput DBS serology for anti-spike antibodies is feasible as an additional test that can be performed using the same sample preparation as the anti-nucleocapsid analysis. The quantitative measurements obtained are accurate enough to track the dynamics of antibody levels in populations, even after vaccination campaigns.

Spatiotemporal Changes of Antibiotic Resistance, Potential Pathogens, and Health Risk in Kindergarten Dust.

The microorganisms present in kindergartens are extremely important for children's health during their three-year preschool education. To assess the risk of outdoor dust in kindergartens, the antibiotic resistome and potential pathogens were investigated in dust samples collected from 59 kindergartens in Xiamen, southeast China in both the winter and summer. Both high-throughput quantitative PCR and metagenome analysis revealed a higher richness and abundance of antibiotic resistance genes (ARGs) in winter (P < 0.05). Besides, the bloom of ARGs and potential pathogens was evident in the urban kindergartens. The co-occurrence patterns among ARGs, mobile genetic elements (MGEs), and potential pathogens suggested some bacterial pathogens were potential hosts of ARGs and MGEs. We found a large number of high-risk ARGs in the dust; the richness and abundance of high-risk ARGs were higher in winter and urban kindergartens compared to in summer and peri-urban kindergartens, respectively. The results of the co-occurrence patterns and high-risk ARGs jointly reveal that urbanization will significantly increase the threat of urban dust to human beings and their risks will be higher in winter. This study unveils the close association between ARGs/mobile ARGs and potential pathogens and emphasizes that we should pay more attention to the health risks induced by their combination.

High-Throughput Quantitative Analysis of Amino Acids in Freeze-Dried Drops Using Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become a promising analytical tool for molecular profiling in biological applications. However, its ultrahigh vacuum environment and matrix effects hamper the absolute quantitation of solution samples. Herein, we present a rapid high-throughput platform for quantitative ToF-SIMS analysis of amino acids in matrix deposits formed from freeze-dried solution drops through ice sublimation on a parylene film microarray substrate. Droplets of the amino acid solutions, which were mixed with stable isotope-labeled phenylalanine (F*) of high concentration (10 mM), were loaded on wells of the microarray, then frozen and evaporated slowly below the freezing point, forming continuous solid-phase F* matrix deposits. The amino acids (≤500 μM), adequately well dispersed throughout the F* matrix deposits on each well, were quantitatively analyzed by ToF-SIMS in a rapid and high-throughput fashion. The lower limit of quantitation reached below 10 μM.

Intramolecular Ring-Chain Equilibrium Elimination Strategy for Pinpointing C═C Positional and Geometric Isomers of N-Alkylpyridinium Unsaturated Fatty Acid Derivatives via Ion Mobility-Mass Spectrometry.

Free unsaturated fatty acids (UFA) are key intermediates of lipid metabolism and participate in many metabolic pathways with specific biological functions. Although various fragmentation-based methods for pinpointing C═C locations in UFA were developed, the current mass spectrometry methods are difficult to simultaneously differentiate geometric isomers and positional isomers in trace samples due to low ionization efficiency, low conversion, and low resolution. Herein, an intramolecular ring-chain equilibrium elimination strategy via 4-plex stable isotope labeling dual derivatization-assisted ion mobility-mass spectrometry was developed, thereby one-pot specifically labeling C═C and carboxyl groups among the trace and unstable UFA with high sensitivity, high efficiency, and good substrate generality. It achieved fast separation of both C═C positional and geometric isomers with high resolution, which benefited from eliminating the intramolecular ring-chain equilibrium by suppressing the formation of salt bridges between free carboxyl groups and pyridine cations. 4-plex stable isotope labeling reagents showed similar reactivity, enabling high-throughput quantitative analysis of omics. This method was successfully applied for accurate and rapid identification of the UFA composition in olive oil extract. These results suggest that the developed method provides new insight for rapid characterization of UFA C═C positional and geometric isomers in complex samples to explore disease biomarkers and food quality control indicators.

Astragaloside IV Alleviates Acute Liver Failure Induced by D-GalN/LPS by Upregulating Autophagy and Reducing Inflammation

Abstract Acute liver failure (ALF) is a life-threatening condition that manifests in an extremely serious manner and progresses rapidly. The following study investigated the protective effect of astragaloside IV (AS-IV), a traditional Chinese drug, on ALF, and its underlying mechanisms, focusing on autophagy and inflammation regulation. Mice were randomly divided into a saline group, a D-galactosamine and lipopolysaccharide (D-GalN/LPS) group and an AS-IV group. Biochemical analysis, immunohistochemistry, cytometric bead array, high-throughput quantitative PCR, flow cytometry and Western analysis were used to assess inflammation and liver damage 5 hours after D-GalN/LPS exposure. Astragaloside IV treatment reduced mortality by alleviating D-GalN/LPS–induced hepatic damage and decreasing inflammation (decreasing Ly6c+ monocyte levels, reducing inflammatory cytokines and increasing anti-inflammatory factors) as well as upregulating autophagy. Furthermore, PCR array was used to detect expression of autophagy-related genes, which demonstrated a Log2 fold change in gene expression between the AS-IV and D-GalN/LPS groups ranging from 1.19 to −3.53, with Tnfsf10 showing the largest alteration between the two groups. These data suggest that AS-IV may alleviate ALF by upregulating autophagy and reducing inflammation, and it may therefore be an interesting drug for alleviating ALF.

12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.

Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC-MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.

A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein.

Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

Determining the spatiotemporal variation, sources, and ecological processes of antibiotic resistance genes in a typical lake of the middle reaches of the Yangtze River

Antibiotic resistance genes (ARGs) are emerging environmental pollutants, influenced by complex regulatory factors. River-lake systems act as natural reservoirs for ARGs and provide an ideal model for studying their regulatory mechanisms. This study employed high-throughput quantitative PCR, high-throughput sequencing, correlation analyses, and model predictions to investigate the dynamics of ARGs and their influencing factors in Liangzi Lake, located in the mid-reaches of the Yangtze River. The research specifically centered on three environmental components: lake water, sediment, and river water. Results indicated that the ARGs from eight major antibiotic classes, displaying distinct seasonal distribution patterns. In comparison to the sediment, the water phase demonstrated a higher diversity of ARGs, with the highest level of ARGs sharing observed between lake and river waters (approximately 83.7 %). Furthermore, seasonal variations significantly influenced the distributions of both ARGs and bacterial communities. The diversity of ARGs was highest during the summer and autumn, and specific bacterial species exhibited robust correlations with ARGs (including matA/mel, aac (6′)-Ib-03, and blaROB). It is worth noting that environmental attributes and bacterial diversity had the most substantial impact on the dynamic changes in ARGs. Lastly, source tracking analysis pinpointed that sediment as the primary source of ARGs in lake water, constituting 45 % to 48 % of the total ARGs. Our study provides a comprehensive analysis of ARGs and their influencing factors in the river-lake system of the middle reaches of the Yangtze River, with Liangzi Lake as a representative case.

High-throughput screening and quantitative analysis of hidden ingredients in plant protection products by GC-MS/MS

Hidden ingredients in plant protection products (PPPs) threaten public health, food trade, and the environment. In this study, we developed a high-throughput screening method of 639 hidden ingredients in PPPs using GC-MS/MS in multiple reaction monitoring mode. Results showed that the qualitative criteria of retention time (tR) shift and uncertainty of qualifier to quantifier ratio in the commercially available Shimadzu Smart Pesticides Database were set at < 0.17 min and < 30%, respectively, which could be used to tentatively identify compounds without standards. The limits of quantification were 0.01–0.05 mg/kg. A wide linear range of 10–1000 μg/L was observed with R2 ≥ 0.975. Recoveries from three types of PPP formulations were 62.08%− 126.3%, with relative standard deviations < 15.7%. Finally, this method was applied to screen and quantify hidden ingredients in 91 plant protection products (PPPs) samples collected from online sales in China. Only one hidden ingredient, dimethomorph (1.6 g/kg), was detected in the polyoxin formulation (15% wettable powder). The results will be helpful in assessing the potential risks of hidden ingredients in PPPs.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

To promote the clinical application of PET/MRI in oncology

PET/MRI integrates anatomical, functional and metabolic information, and is increasingly used in the field of clinical oncology, including early diagnosis of disease, local staging, detection of systemic metastases, evaluation of treatment efficacy and prognosis. In addition to fine anatomical structure, MRI multiparametric imaging can also provide functional information of the lesion. Combined with metabolic information of PET, MRI multiparametric imaging can better evaluate the benign and maligant lesions, tumor grade and extent of invasion, and identify tumor recurrence and radiation necrosis. The development of new PET tracers, optimization of MRI sequences and examination paths, combined with high-throughput image quantitative analysis methods, radiomics, artificial intelligence and other new technologies will further promote the application of PET/MRI in oncology.

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.

Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart. Atrial tissue samples from animals with AF reveal an abnormal increase in cardiomyocytes death and alterations in calcium handling proteins. High-throughput quantitative proteomics and immunoblotting analyses at different stages of AF progression identify downregulation of contractile proteins and progressive increase in atrial fibrosis. Moreover, advanced optical mapping techniques, applied to whole heart preparations during AF, demonstrate that AF-related remodeling decreases the frequency threshold for dissociation between transmembrane voltage signals and intracellular calcium transients compared to healthy controls. Single cell simulations of human atrial cardiomyocytes also confirm the experimental results. In patients, non-invasive assessment of the atrial electromechanical relationship further demonstrate that atrial electromechanical dissociation is an early prognostic indicator for acute and long-term rhythm control.

Development of novel isobaric tags enables accurate and sensitive multiplexed proteomics using complementary ions.

High-throughput quantitative analysis of the cells' proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics.

Tools to analyze the organization and formation of the germline cyst in zebrafish oogenesis.

Oocytes develop in the germline cyst, a cellular organization in which germ cells are tightly interconnected and surrounded by somatic cells. The cyst produces oocytes for follicle formation and is a hub for essential processes in meiosis and oocyte differentiation. However, the formation and organization of the cyst, and their contribution to oocyte production in vertebrates remain unclear. Here, we provide tools for three-dimensional and functional in vivo analyses of the germline cyst in the zebrafish ovary. We describe the use of serial block-face scanning electron microscopy (SBF-SEM) to resolve the three-dimensional architecture of cells and organelles in the cyst at ultrastructural resolution. We present a deep learning-based pipeline for high-throughput quantitative analysis of three-dimensional confocal datasets of cysts in vivo. We provide a method for laser ablation of cellular components for manipulating cyst cells in ovaries. These methods will facilitate the investigation of the cyst cellular organization, expand the toolkit for the study of the zebrafish ovary, and advance our understanding of female developmental reproduction. They could also be further applied to the investigation of other developmental systems.

Bubble-free diatoms polymerase chain reaction

Polymerase chain reaction (PCR) in small fluidic systems not only improves speed and sensitivity of deoxyribonucleic acid (DNA) amplification but also achieves high-throughput quantitative analyses. However, air bubble trapping and growth during PCR has been considered as a critical problem since it causes the failure of DNA amplification. Here we report bubble-free diatom PCR by exploiting a hierarchically porous silica structure of single-celled algae. We show that femtoliters of PCR solution can be spontaneously loaded into the diatom interior without air bubble trapping due to the surface hydrophilicity and pore structure of the diatom. We discover that a large pressure gradient between air bubbles and nanopores rapidly removes residual air bubbles through the periodically arrayed nanopores during thermal cycling. We demonstrate the DNA amplification by diatom PCR without air bubble trapping and growth. Finally, we successfully detect DNA fragments of SARS-CoV-2 with as low as 10 copies/μl by devising a microfluidic device integrated with diatoms assembly. We believe that our work can be applied to many PCR applications for innovative molecular diagnostics and provides new opportunities for naturally abundant diatoms to create innovative biomaterials in real-world applications.