Mechanistic insight into glioma through spatially multidimensional proteomics.

Characterizing the tumor microenvironment at the molecular level is essential for understanding the mechanisms of tumorigenesis and evolution. However, the specificity of the blood proteome in localized region of the tumor and its linkages with other systems is difficult to investigate. Here, we propose a spatially multidimensional comparative proteomics strategy using glioma as an example. The blood proteome signature of tumor microenvironment was specifically identified by in situ collection of arterial and venous blood from the glioma region of the brain for comparison with peripheral blood. Also, by integrating with different dimensions of tissue and peripheral blood proteomics, the information on the genesis, migration, and exchange of glioma-associated proteins was revealed, which provided a powerful method for tumor mechanism research and biomarker discovery. The study recruited multidimensional clinical cohorts, allowing the proteomic results to corroborate each other, reliably revealing biological processes specific to gliomas, and identifying highly accurate biomarkers.

The genetic etiology of body fluids on chronic obstructive airways disease.

BACKGROUND: Numerous studies have documented significant alterations in the bodily fluids of Chronic Obstructive Pulmonary Disease (COPD) patients. However, existing literature lacks causal inference due to residual confounding and reverse causality. METHODS: Summary-level data for COPD were obtained from two national biobanks: the UK Biobank, comprising 1,605 cases and 461,328 controls, and FinnGen, with 6,915 cases and 186,723 controls. We also validated our findings using clinical data from 2,690 COPD patients and 3,357 healthy controls from the First Affiliated Hospital of Guangzhou Medical University. A total of 44 bodily fluid biomarkers were selected as candidate risk factors. Mendelian randomization (MR) and meta-analyses were used to evaluate the causal effects of these bodily fluids on COPD and lung function (FEV1/FVC). RESULTS: Mendelian randomization (MR) and meta-analyses, by integrating data from the UK Biobank and FinnGen cohort, found that 3 bodily fluids indicators (HDLC, EOS, and TP) were causally associated with the risk of COPD, two (EOS and TP) of which is consistent with our observational findings. Moreover, we noticed EOS and TP were causally associated with the risk of lung function (FEV1/FVC). CONCLUSIONS: The MR findings and clinical data highlight the independent and significant roles of EOS and TP in the development of COPD and lung function (FEV1/FVC), which might provide a deeper insight into COPD risk factors and supply potential preventative strategies.

Impact of ultra-low temperature storage on serum sIgE detection and allergic disease biobank feasibility.

Research has shown that the concentration and composition of biological samples may change after long-term ultra-low temperature storage. Consequently, this study examined the effect of ultra-low temperature storage on serum sIgE detection by comparing sIgE concentrations at various durations from the time of sample storage to subsequent testing. We selected 40 serum samples from the Guangzhou Medical University Affiliated First Hospital Biobank, which had been tested for house dust mites, dog hair, tobacco mold, cockroaches, and cow milk allergen sIgE. Samples were categorized by storage duration: 14 samples stored for 10 years, 12 for 5 years, and 14 for 3 years. They were also classified by sIgE positive levels: 15 samples at levels 1-2, 15 at levels 3-4, and 10 at levels 5-6. The allergen sIgE of these samples was retested using the same technology. Regardless of the type of allergen or the level of positivity, the majority of sIgE concentrations measured at the time of storage were higher than the current measurements, but the difference was not statistically significant. The correlation between the sIgE results at the time of storage and the current results was high for samples stored for 10 years (rs = 0.991, P < 0.001) and 5 years (rs = 0.964, P < 0.001). Serum allergen sIgE is stable when stored under ultra-low temperature conditions, making the construction of a biological sample bank for allergic diseases feasible. This will facilitate researchers in quickly obtaining samples, conducting technical research, and translating findings, thereby promoting the development of the field of allergy through integration of industry, academia, and research.

Ultra-high-throughput mapping of the chemical space of asymmetric catalysis enables accelerated reaction discovery.

The discovery of highly enantioselective catalysts and elucidating their generality face great challenges due to the complex multidimensional chemical space of asymmetric catalysis and inefficient screening methods. Here, we develop a general strategy for ultra-high-throughput mapping of the chemical space of asymmetric catalysis by escaping the time-consuming chiral chromatography separation. The ultrafast ( ~ 1000 reactions/day) and accurate (median error < ±1%) analysis of enantiomeric excess are achieved through the ion mobility-mass spectrometry combines with the diastereoisomerization strategy. A workflow for accelerated asymmetric reaction screening is established and verified by mapping the large-scale chemical space of more than 1600 reactions of α-asymmetric alkylation of aldehyde with organocatalysis and photocatalysis. Importantly, a class of high-enantioselectivity primary amine organocatalysts of 1,2-diphenylethane-1,2-diamine-based sulfonamides is discovered by the accelerated screening, and the mechanism for high-selectivity is demonstrated by computational chemistry. This study provides a practical and robust solution for large-scale screening and discovery of asymmetric reactions.

Elucidating the Underlying Reactivities of Alternating Current Electrosynthesis by Time-Resolved Mapping of Short-Lived Reactive Intermediates.

Alternating current (AC) electrolysis is an emerging field in synthetic chemistry, however its mechanistic studies are challenged by the effective characterization of the elusive intermediate processes. Herein, we develop an operando electrochemical mass spectrometry platform that allows time-resolved mapping of stepwise electrosynthetic reactive intermediates in both direct current and alternating current modes. By dissecting the key intermediate processes of electrochemical functionalization of arylamines, the unique reactivities of AC electrosynthesis, including minimizing the over-oxidation/reduction through the inverse process, and enabling effective reaction of short-lived intermediates generated by oxidation and reduction in paired electrolysis, were evidenced and verified. Notably, the controlled kinetics of reactive N-centered radical intermediates in multistep sequential AC electrosynthesis to minimize the competing reactions was discovered. Overall, this work provides direct evidence for the mechanism of AC electrolysis, and clarifies the underlying reasons for its high efficiency, which will benefit the rational design of AC electrosynthetic reactions.

Nontargeted metabolomics analysis of follicular fluid in patients with endometriosis provides a new direction for the study of oocyte quality.

Endometriosis is a common, estrogen-dependent chronic gynecological disease that endangers the reproductive system and systemic metabolism of patients. We aimed to investigate the differences in metabolic profiles in the follicular fluid between infertile patients with endometriosis and controls. A total of 25 infertile patients with endometriosis and 25 infertile controls who were similar in age, BMI, fertilization method and ovulation induction treatment were recruited in this study. Metabolomics analysis of follicular fluid was performed by two methods of high-performance liquid chromatography tandem mass spectrometry. There were 36 upregulated and 17 downregulated metabolites in the follicular fluid of patients in the endometriosis group. KEGG pathway analysis revealed that these metabolites were enriched in phenylalanine, tyrosine and tryptophan biosynthesis, aminoacyl-tRNA biosynthesis, phenylalanine metabolism and pyrimidine metabolism pathways. A biomarker panel consisting of 20 metabolites was constructed by random forest, with an accuracy of 0.946 and an AUC of 0.988. This study characterizes differences in follicular fluid metabolites and associated pathway profiles in infertile patients with endometriosis. These findings can provide a better comprehensive understanding of the disease and a new direction for the study of oocyte quality, as well as potential metabolic markers for the prognosis of endometriosis.

Lipidomic Characterization of Oocytes at Single-Cell Level Using Nanoflow Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Mass spectrometry (MS)-based lipidomic has become a powerful tool for studying lipids in biological systems. However, lipidome analysis at the single-cell level remains a challenge. Here, we report a highly sensitive lipidomic workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS)-MS. This approach enables the high-coverage identification of lipidome landscape at the single-oocyte level. By using the proposed method, comprehensive lipid changes in porcine oocytes during their maturation were revealed. The results provide valuable insights into the structural changes of lipid molecules during porcine oocyte maturation, highlighting the significance of sphingolipids and glycerophospholipids. This study offers a new approach to the single-cell lipidomic.

High-Coverage Four-Dimensional Data-Independent Acquisition Proteomics and Phosphoproteomics Enabled by Deep Learning-Driven Multidimensional Predictions.

Four-dimensional (4D) data-independent acquisition (DIA)-based proteomics is a promising technology. However, its full performance is restricted by the time-consuming building and limited coverage of a project-specific experimental library. Herein, we developed a versatile multifunctional deep learning model Deep4D based on self-attention that could predict the collisional cross section, retention time, fragment ion intensity, and charge state with high accuracies for both the unmodified and phosphorylated peptides and thus established the complete workflows for high-coverage 4D DIA proteomics and phosphoproteomics based on multidimensional predictions. A 4D predicted library containing ∼2 million peptides was established that could realize experimental library-free DIA analysis, and 33% more proteins were identified than using an experimental library of single-shot measurement in the example of HeLa cells. These results show the great values of the convenient high-coverage 4D DIA proteomics methods.

Metabonomic analysis of follicular fluid in patients with diminished ovarian reserve.

Background: Ovarian reserve is an important factor determining female reproductive potential. The number and quality of oocytes in patients with diminished ovarian reserve (DOR) are reduced, and even if in vitro fertilization-embryo transfer (IVF-ET) is used to assist their pregnancy, the clinical pregnancy rate and live birth rate are still low. Infertility caused by reduced ovarian reserve is still one of the most difficult clinical problems in the field of reproduction. Follicular fluid is the microenvironment for oocyte survival, and the metabolic characteristics of follicular fluid can be obtained by metabolomics technology. By analyzing the metabolic status of follicular fluid, we hope to find the metabolic factors that affect the quality of oocytes and find new diagnostic markers to provide clues for early detection and intervention of patients with DOR. Methods: In this research, 26 infertile women with DOR and 28 volunteers with normal ovarian reserve receiving IVF/ET were recruited, and their follicular fluid samples were collected for a nontargeted metabonomic study. The orthogonal partial least squares discriminant analysis model was used to understand the separation trend of the two groups, KEGG was used to analyze the possible metabolic pathways involved in differential metabolites, and the random forest algorithm was used to establish the diagnostic model. Results: 12 upregulated and 32 downregulated differential metabolites were detected by metabolic analysis, mainly including amino acids, indoles, nucleosides, organic acids, steroids, phospholipids, fatty acyls, and organic oxygen compounds. Through KEGG analysis, these metabolites were mainly involved in aminoacyl-tRNA biosynthesis, tryptophan metabolism, pantothenate and CoA biosynthesis, and purine metabolism. The AUC value of the diagnostic model based on the top 10 metabolites was 0.9936. Conclusion: The follicular fluid of patients with DOR shows unique metabolic characteristics. These data can provide us with rich biochemical information and a research basis for exploring the pathogenesis of DOR and predicting ovarian reserve function.

Dual-resolving of positional and geometric isomers of C=C bonds via bifunctional photocycloaddition-photoisomerization reaction system.

The biological functions of lipids largely depend on their chemical structures. The position and configuration of C=C bonds are two of the essential attributes that determine the structures of unsaturated lipids. However, simultaneous identification of both attributes remains challenging. Here, we develop a bifunctional visible-light-activated photocycloaddition-photoisomerization reaction system, which enables the dual-resolving of the positional and geometric isomerism of C=C bonds in lipids when combines with liquid chromatography-mass spectrometry. The dual-pathway reaction mechanism is demonstrated by experiments and density functional theory calculations. Based on this bifunctional reaction system, a workflow of deep structural lipidomics is established, and allows the revealing of unique patterns of cis-trans-isomers in bacteria, as well as the tracking of C=C positional isomers changes in mouse brain ischemia. This study not only offers a powerful tool for deep lipid structural biology, but also provides a paradigm for developing the multifunctional visible-light-induced reaction.

Machine Learning of Serum Metabolic Patterns Encodes Asymptomatic SARS-CoV-2 Infection.

Asymptomatic COVID-19 has become one of the biggest challenges for controlling the spread of the SARS-CoV-2. Diagnosis of asymptomatic COVID-19 mainly depends on quantitative reverse transcription PCR (qRT-PCR), which is typically time-consuming and requires expensive reagents. The application is limited in countries that lack sufficient resources to handle large-scale assay during the COVID-19 outbreak. Here, we demonstrated a new approach to detect the asymptomatic SARS-CoV-2 infection using serum metabolic patterns combined with ensemble learning. The direct patterns of metabolites and lipids were extracted by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) within 1 s with simple sample preparation. A new ensemble learning model was developed using stacking strategy with a new voting algorithm. This approach was validated in a large cohort of 274 samples (92 asymptomatic COVID-19 and 182 healthy control), and provided the high accuracy of 93.4%, with only 5% false negative and 7% false positive rates. We also identified a biomarker panel of ten metabolites and lipids, as well as the altered metabolic pathways during asymptomatic SARS-CoV-2 Infection. The proposed rapid and low-cost approach holds promise to apply in the large-scale asymptomatic COVID-19 screening.

High-Throughput Nano-Electrostatic-Spray Ionization/Photoreaction Mass Spectrometric Platform for the Discovery of Visible-Light-Activated Photocatalytic Reactions in the Picomole Scale.

Visible-light-activated photocatalysis has emerged as a green and powerful tool for the synthesis of various organic compounds under mild conditions. However, the expeditious discovery of novel photocatalysts and synthetic pathways remains challenging. Here, we developed a bifunctional platform that enabled the high-throughput discovery and optimization of new photochemical reactions down to the picomole scale. This platform was designed based on a contactless nano-electrostatic-spray ionization technique, which allows synchronized photoreactions and high-throughput in situ mass spectrometric analysis with a near-100% duty cycle. Using this platform, we realized the rapid screening of photocatalytic reactions in ambient conditions with a high speed of less than 1.5 min/reaction using picomolar materials. The versatility was validated by multiple visible-light-induced photocatalytic reactions, especially the discovery of aerobic C-H thiolation with low-cost organic photocatalysts without any other additives. This study provided a new paradigm for the integration of ambient ionization techniques and new insights into photocatalytic reaction screening, which will have broad applications in the development of new visible-light-promoted reactions.

Distinct lipid metabolic dysregulation in asymptomatic COVID-19.

Asymptomatic infection is a big challenge in curbing the spread of COVID-19. However, its identification and pathogenesis elucidation remain issues. Here, by performing comprehensive lipidomic characterization of serum samples from 89 asymptomatic COVID-19 patients and 178 healthy controls, we screened out a panel of 15 key lipids that could accurately identify asymptomatic patients using a new ensemble learning model based on stacking strategy with a voting algorithm. This strategy provided a high accuracy of 96.0% with only 3.6% false positive rate and 4.8% false negative rate. More importantly, the unique lipid metabolic dysregulation was revealed, especially the enhanced synthesis of membrane phospholipids, altered sphingolipids homeostasis, and differential fatty acids metabolic pattern, implicating the specific host immune, inflammatory, and antiviral responses in asymptomatic COVID-19. This study provides a potential prediagnostic method for asymptomatic COVID-19 and molecular clues for the pathogenesis and therapy of this disease.

Reactive Olfaction Ambient Mass Spectrometry.

Chemical ionization of organic compounds with negligible vapor pressures (VP) is achieved at atmospheric pressure when the proximal sample is exposed to corona discharge. The vapor-phase analyte is produced through a reactive olfaction process, which is determined to include electrostatic charge induction in the proximal condensed-phase sample, resulting in the liberation of free particles. With no requirement for physical contact, a new contained nano-atmospheric pressure chemical ionization (nAPCI) source was developed that allowed direct mass spectrometry analysis of complex mixtures at a sample consumption rate less than nmol/min. The contained nAPCI source was applied to analyze a wide range of samples including the detection of 1 ng/mL cocaine in serum and 200 pg/mL caffeine in raw urine, as well as the differentiation of chemical composition of perfumes and beverages. Polar (e.g., carminic acid; estimated VP 5.1 × 10-25 kPa) and nonpolar (e.g., vitamin D2; VP 8.5 × 10-11 kPa) compounds were successfully ionized by the contained nAPCI ion source under ambient conditions, with the corresponding ion types of 78 other organic compounds characterized.

An integrated mass spectrometry platform enables picomole-scale real-time electrosynthetic reaction screening and discovery.

The identification of new electrosynthetic pathways enables environmentally friendly synthetic applications. However, the development of miniaturized screening procedures/platforms to expedite the discovery of electrooxidation reactions remains challenging. Herein, we developed an integrated system that serves as a reactor and ion source in a single experimental step using only picomole-scale reactants to monitor electrooxidation in real-time. This reaction screening platform utilizes the intrinsic electrochemical capabilities of nano-electrospray ionization mass spectrometry. We validated the feasibility of this method by reproducing three known electrochemical reactions. We also discovered two new electroorganic reaction pathways: (i) C-N dehydrodimerization of 8-methyl-1,2,3,4-tetrahydroquinoline to construct a novel quinoline skeleton, and (ii) TEMPO-mediated accelerated electrooxidative dehydrogenation of tetrahydroisoquinolines. Moreover, the radical cations and key intermediates captured by this screening platform provided direct evidence for the mechanism of these novel electrochemical reactions.

Picomole-Scale Real-Time Photoreaction Screening: Discovery of the Visible-Light-Promoted Dehydrogenation of Tetrahydroquinolines under Ambient Conditions.

The identification of new photocatalytic pathways expands our knowledge of chemical reactivity and enables new environmentally friendly synthetic applications. However, the development of miniaturized screening procedures/platforms to expedite the discovery of photochemical reactions remains challenging. Herein, we describe a picomole-scale, real-time photoreaction screening platform in which a handheld laser source is coupled with nano-electrospray ionization mass spectrometry. By using this method, we discovered an accelerated dehydrogenation pathway for the conversion of tetrahydroquinolines into the corresponding quinolines. This transformation is readily promoted by an off-the-shelf [Ru(bpy)3 ]Cl2 ⋅6 H2 O complex in air at ambient temperature in direct sunlight, or with the aid of an energy-saving lamp. Moreover, radical cations and trans-dihydride intermediates captured by the screening platform provided direct evidence for the mechanism of the photoredox reaction.

Mass Spectrometry for Paper-Based Immunoassays: Toward On-Demand Diagnosis.

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Mass spectrometry imaging reveals the sub-organ distribution of carbon nanomaterials.

Label and label-free methods to image carbon-based nanomaterials exist. However, label-based approaches are limited by the risk of tag detachment over time, and label-free spectroscopic methods have slow imaging speeds, weak photoluminescence signals and strong backgrounds. Here, we present a label-free mass spectrometry imaging method to detect carbon nanotubes, graphene oxide and carbon nanodots in mice. The large molecular weights of nanoparticles are difficult to detect using conventional mass spectrometers, but our method overcomes this problem by using the intrinsic carbon cluster fingerprint signal of the nanomaterials. We mapped and quantified the sub-organ distribution of the nanomaterials in mice. Our results showed that most carbon nanotubes and nanodots were found in the outer parenchyma of the kidney, and all three materials were seen in the red pulp of the spleen. The highest concentrations of nanotubes in the spleen were found within the marginal zone.

Lysosomal pH rise during heat shock monitored by a lysosome-targeting near-infrared ratiometric fluorescent probe.

Heat stroke is a life-threatening condition, featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood, mainly because of the lack of a suitable research approach. Herein, by incorporating morpholine into a stable hemicyanine skeleton, we develop a new lysosome-targeting near-infrared ratiometric pH probe. In combination with fluorescence imaging, we show for the first time that the lysosomal pH value increases but never decreases during heat shock, which might result from lysosomal membrane permeabilization. We also demonstrate that this lysosomal pH rise is irreversible in living cells. Moreover, the probe is easy to synthesize, and shows superior overall analytical performance as compared to the existing commercial ones. This enhanced performance may enable it to be widely used in more lysosomal models of living cells and in further revealing the mechanisms underlying heat-related pathology.

A cresyl violet-based fluorescent off-on probe for the detection and imaging of hypoxia and nitroreductase in living organisms.

A new cresyl violet-based fluorescent off-on probe has been developed through a one-step synthesis for the detection of nitroreductase (NTR) and hypoxia. The detection mechanism is based on the NTR-catalyzed reduction of the probe to cresyl violet, accompanied with a large fluorescence enhancement at a long wavelength of 625 nm. The probe can detect NTR in aqueous solution with high selectivity and sensitivity, and the detection limit is 1 ng mL(-1) NTR. Most importantly, the probe has been successfully used to image not only NTR and hypoxia in living cells, but also the distribution of NTR in zebrafish in vivo.