Mass Spectrometry Research Articles

Femtosecond Laser Assisted Chemical Ionization Mass Spectrometry: Toward Sub-ppq Detection Limits for Organic Molecules.

Ultrasensitive analysis of organic molecules is crucial for various fundamental research and applications. State-of-the-art techniques for this purpose can achieve detection limits of several hundred ppq (parts-per-quadrillion), while a higher sensitivity is pursued constantly. To achieve this goal, we develop femtosecond laser assisted chemical ionization for mass spectrometry. This technique combines the advantages of femtosecond laser ionization and chemical ionization, either of which enables subppt (parts-per-trillion) mass spectrometry analysis of organic molecules. The results demonstrate that the developed ionization technique, when employed in mass spectrometry, can surpass femtosecond laser ionization by more than 3 orders of magnitude in sensitivity, while still maintaining good versatility and the ability to work under ambient conditions. This work paves the way for subppq analysis of organic molecules in gas phase and even in ambient environments, which can open up new research fields for trace substance analysis in atmospheric environment, clinical diagnosis, biomedical studies, etc.

Proteomic Characterization of Urinary Exosomes with Pancreatic Cancer by Phosphatidylserine Imprinted Polymer Enrichment and Mass Spectrometry Analysis.

Exosomes, as carriers of cell-to-cell communication, can serve as promising biomarkers for probing the early diagnosis of cancer. Pancreatic cancer is a common malignant tumor of the pancreas with an insidious onset and difficult early diagnosis. The aim of this study was to capture exosomes in urine samples by phosphatidylserine-molecularly imprinted polymers (PS-MIPs). Transmission electron microscopy and nanoparticle tracking analysis as well as Western blot showed that our molecularly imprinted material can effectively capture urinary exosomes. Three parallel tests verified the reproducibility of the mass spectrometry assay and the stability of the material capture efficiency. Mass Spectrometry with nontargeted proteomics was combined to show differentially expressed proteins in exosomes between 5 pancreatic cancer patients and 5 healthy controls. The most significant changes in the proteomic profile in pancreatic cancer patients compared to healthy controls were the overexpression of SLC9A3R1, SPAG9, and ferritin light chain (FTL) These proteins may have an important role in diagnosis and prognostic assessment, supporting further scientific and clinical studies on pancreatic cancer.

A single-center study on the distribution and shifting trend of infectious keratitis pathogens from 2018 to 2022

Objective: To investigate the pathogen species, composition, and distribution characteristics of infectious keratitis pathogens in Shandong Province and its surrounding areas. Methods: In this cross-sectional study, patients with keratitis who underwent corneal sampling and microbiological culture at the Shandong Eye Hospital from January 1, 2018 to December 31, 2022 were included. Under topical anesthesia, the edge of the lesion was scraped by an experienced physician. The samples were inoculated on blood agar and Sabouraud dextrose agar plates, separately for bacterial and fungal culture and identification. If necessary, the samples were inoculated on a non-nutrient agar medium with Escherichia coli for Acanthamoeba culture. Bacterial isolates were identified using Vitek 2 compact or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fungal isolates were identified based on morphological characteristics or sent to a company for sequencing in cases of difficult identification. The epidemiological data of the patients, pathogen species and number (counting as 1 strain if the same strain was isolated from multiple corneal specimens of the same patient), culture positivity rate, and seasonal distribution were recorded. Differences in pathogen positivity rates among different seasons were analyzed using the chi-square test. Results: Among the 4, 024 patients with infectious keratitis during the study period, there were 2 510 males (62.3%) and 1 514 females (37.6%), aged from 46 days to 94 years. Positive microbial culture results were achieved in 2, 363 patients (58.7%), including 906 cases (38.3%) with bacterial positivity, 1 231 cases (52.1%) with fungal positivity, 28 cases (1.2%) with Acanthamoeba positivity, and 198 cases (8.4%) with mixed fungal and bacterial infections. A total of 2 561 strains were isolated, including 1 104 bacterial strains. The most common bacteria were coagulase-negative Staphylococcus spp. (623/1 104, 56.4%), followed by Streptococcus spp. (131/1 104, 11.9%) and Pseudomonas aeruginosa (68/1 104, 6.2%). The most common fungi were Fusarium spp. (634/1 429, 44.4%), followed by Aspergillus spp. (279/1 429, 19.5%) and Alternaria spp. (229/1 429, 16.0%). Bacterial keratitis was more common in summer and autumn, while fungal keratitis was more common in autumn and winter. Conclusions: Among infectious keratitis cases in Shandong Eye Hospital, Fusarium species were predominant fungal pathogens, while coagulase-negative Staphylococcus predominated in bacterial pathogens. Both fungal and bacterial corneal infections showed seasonal variations.

Recent Advances in Monitoring Microbial Toxins in Food Samples by HPLC-Based Techniques: A Review

This study examines the significant impact of bacterial, algal, and fungal toxins on foodborne illnesses, and stresses the importance of advanced detection techniques, such as high-performance liquid chromatography (HPLC)-based methodologies. It emphasizes the urgent need for further advancements in these techniques to ensure food safety, as they offer significant benefits, including low detection limits and the ability to be combined with other techniques to detect a wide range of toxins. In this regard, HPLC has emerged as a versatile and sensitive analytical technique for this purpose. Various HPLC methods, often enhanced with detectors such as ultraviolet (UV), fluorescence (FD), and mass spectrometry (MS), have been developed to identify and quantify microbial toxins in a wide variety of food samples. Recent advancements include HPLC-FD methods that utilize the natural fluorescence of certain aflatoxins, improving detection sensitivity. HPLC-MS/MS and UHPLC-MS/MS techniques offer high selectivity and sensitivity, making them suitable for detecting a wide range of toxins in trace quantities. The adaptability of HPLC, combined with innovative detection technologies and sample preparation methods, holds significant potential for enhancing food safety monitoring and reducing the global burden of foodborne diseases.

Nesfatin-1 enhances vascular smooth muscle calcification through facilitating BMP-2 osteogenic signaling

Vascular calcification (VC) arises from the accumulation of calcium salts in the intimal or tunica media layer of the aorta, contributing to higher risk of cardiovascular events and mortality. Despite this, the mechanisms driving VC remain incompletely understood. We previously described that nesfatin-1 functioned as a switch for vascular smooth muscle cells (VSMCs) plasticity in hypertension and neointimal hyperplasia. In this study, we sought to investigate the role and mechanism of nesfatin-1 in VC. The expression of nesfatin-1 was measured in calcified VSMCs and aortas, as well as in patients. Loss- and gain-of-function experiments were evaluated the roles of nesfatin-1 in VC pathogenesis. The transcription activation of nesfatin-1 was detected using a mass spectrometry. We found higher levels of nesfatin-1 in both calcified VSMCs and aortas, as well as in patients with coronary calcification. Loss-of-function and gain-of-function experiments revealed that nesfatin-1 was a key regulator of VC by facilitating the osteogenic transformation of VSMCs. Mechanistically, nesfatin-1 promoted the de-ubiquitination and stability of BMP-2 via inhibiting the E3 ligase SYTL4, and the interaction of nesfatin-1 with BMP-2 potentiated BMP-2 signaling and induced phosphorylation of Smad, followed by HDAC4 phosphorylation and nuclear exclusion. The dissociation of HDAC4 from RUNX2 elicited RUNX2 acetylation and subsequent nuclear translocation, leading to the transcription upregulation of OPN, a critical player in VC. From a small library of natural compounds, we identified that Curculigoside and Chebulagic acid reduced VC development via binding to and inhibiting nesfatin-1. Eventually, we designed a mass spectrometry-based DNA-protein interaction screening to identify that STAT3 mediated the transcription activation of nesfatin-1 in the context of VC. Overall, our study demonstrates that nesfatin-1 enhances BMP-2 signaling by inhibiting the E3 ligase SYTL4, thereby stabilizing BMP-2 and facilitating the downstream phosphorylation of SMAD1/5/9 and HDAC4. This signaling cascade leads to RUNX2 activation and the transcriptional upregulation of MSX2, driving VC. These insights position nesfatin-1 as a potential therapeutic target for preventing or treating VC, advancing our understanding of the molecular mechanisms underlying this critical cardiovascular condition.

Singapore grouper iridovirus VP12 evades the host antiviral immune response by targeting the cGAS-STING signalling pathway.

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus. Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

Microbiome-derived metabolites in early to mid-pregnancy and risk of gestational diabetes: a metabolome-wide association study

BackgroundPre-diagnostic disturbances in the microbiome-derived metabolome have been associated with an increased risk of diabetes in non-pregnant populations. However, the roles of microbiome-derived metabolites, the end-products of microbial metabolism, in gestational diabetes (GDM) remain understudied. We examined the prospective association of microbiome-derived metabolites in early to mid-pregnancy with GDM risk in a diverse population.MethodsWe conducted a prospective discovery and validation study, including a case–control sample of 91 GDM and 180 non-GDM individuals within the multi-racial/ethnic The Pregnancy Environment and Lifestyle Study (PETALS) as the discovery set, a random sample from the PETALS (42 GDM, 372 non-GDM) as validation set 1, and a case–control sample (35 GDM, 70 non-GDM) from the Gestational Weight Gain and Optimal Wellness randomized controlled trial as validation set 2. We measured untargeted fasting serum metabolomics at gestational weeks (GW) 10–13 and 16–19 by gas chromatography/time-of-flight mass spectrometry (TOF–MS), liquid chromatography (LC)/quadrupole TOF–MS, and hydrophilic interaction LC/quadrupole TOF–MS. GDM was diagnosed using the 3-h, 100-g oral glucose tolerance test according to the Carpenter-Coustan criteria around GW 24–28.ResultsAmong 1362 annotated compounds, we identified 140 of gut microbiome metabolism origin. Multivariate enrichment analysis illustrated that carbocyclic acids and branched-chain amino acid clusters at GW 10–13 and the unsaturated fatty acids cluster at GW 16–19 were positively associated with GDM risk (FDR < 0.05). At GW 10–13, the prediction model that combined conventional risk factors and LASSO-selected microbiome-derived metabolites significantly outperformed the model with only conventional risk factors including fasting glucose (discovery AUC: 0.884 vs. 0.691; validation 1: 0.945 vs. 0.731; validation 2: 0.987 vs. 0.717; all P < 0.01). At GW 16–19, similar results were observed (discovery AUC: 0.802 vs. 0.691, P < 0.01; validation 1: 0.826 vs. 0.780; P = 0.10).ConclusionsDysbiosis in microbiome-derived metabolites is present early in pregnancy among individuals progressing to GDM.

NAT10 resolves harmful nucleolar R-loops depending on its helicase domain and acetylation of DDX21

BackgroundAberrant accumulation of R-loops leads to DNA damage, genome instability and even cell death. Therefore, the timely removal of harmful R-loops is essential for the maintenance of genome integrity. Nucleolar R-loops occupy up to 50% of cellular R-loops due to the frequent activation of Pol I transcription. However, the mechanisms involved in the nucleolar R-loop resolution remain elusive. The nucleolar acetyltransferase NAT10 harbors a putative RecD helicase domain (RHD), however, if NAT10 acts in the R-loop resolution is still unknown.MethodsNAT10 knockdown cell lines were constructed using CRISPR/Cas9 technology and short hairpin RNA targeting NAT10 mRNA, respectively. The level of R-loops was detected by immunofluorescent staining combined with RNase H treatment. The helicase activity of NAT10 or DDX21 was determined by in vitro helicase experiment. The interaction between NAT10 and DDX21 was verified by co-immunoprecipitation, immunofluorescent staining and GST pull-down experiments. Acetylation sites of DDX21 by NAT10 were analyzed by mass spectrometry. NAT10 knockdown-induced DNA damage was evaluated by immunofluorescent staining and Western blot detecting γH2AX.ResultsDepletion of NAT10 led to the accumulation of nucleolar R-loops. NAT10 resolves R-loops through an RHD in vitro and in cells. However, Flag-NAT10 ∆RHD mutant still partially reduced R-loop levels in the NAT10-depleted cells, suggesting that NAT10 might resolve R-loops through additional pathways. Further, the acetyltransferase activity of NAT10 is required for the nucleolar R-loop resolution. NAT10 acetylates DDX21 at K236 and K573 to enhance the helicase activity of DDX21 to unwind nucleolar R-loops. The helicase activity of DDX21 significantly decreased by Flag-DDX21 2KR and increased by Flag-DDX21 2KQ in cells and in vitro. Consequently, NAT10 depletion-induced nucleolar R-loop accumulation led to DNA damage, which was rescued by co-expression of Flag-DDX21 2KQ and Flag-NAT10 G641E, demonstrating that NAT10 resolves nucleolar R-loops through bipartite pathways.ConclusionWe demonstrate that NAT10 is a novel R-loop resolvase and it resolves nucleolar R-loops depending on its helicase activity and acetylation of DDX21. The cooperation of NAT10 and DDX21 provides comprehensive insights into the nucleolar R-loop resolution for maintaining genome stability.

Translating 2D-Chromatographic Fingerprinting to Quantitative Volatilomics: Unrevealing Compositional Changes in Maize Silage Volatilome for Robust Marker Discovery.

This study examines the complex volatilome of maize silage, both with and without commercial heterolactic strain inoculation, conserved for 100 days, using quantitative volatilomics. Chemical classes linked to microbial metabolism were analyzed across a concentration range from 10 μg g-1 to 1 ng g-1. A reference method using comprehensive two-dimensional gas chromatography (GC × GC) and time-of-flight mass spectrometry (TOF MS) with loop-type thermal modulation (TM) was translated to a differential-flow modulation (FM) platform with parallel MS and flame ionization detector (FID) detection. With translation, the original method's analyte elution order and resolution are preserved. The new method allowed for accurate quantification using multiple headspace solid-phase microextraction (MHS-SPME) and FID-predicted relative response factors (RRFs). Both methods showed comparable discriminatory power with FM GC × GC-MS/FID achieving satisfactory quantification accuracy without external calibration. Analysis of 98 volatiles provided insights into silage fermentation, supporting marker discovery and correlations with silage quality and stability.

Huangqi-Guizhi-Wuwutang protects against oligospermia in mice by promoting the proliferation of spermatogenic stem cells: A comprehensive study using HPLC-Q-TOF/MS and experimental pharmacology.

The classical traditional Chinese medicine formula Huangqi-Guizhi-Wuwutang (HGW) has been shown to enhance sperm production. However, the bioactive components and comprehensive mechanisms underlying the therapeutic effects remain unclear. The present study investigates the potential active ingredients and underlying mechanisms of HGW against spermatogenesis dysfunction. The chemical components of HGW were analyzed by mass spectrometry. And then the "components-targets-pathway-disease" network was constructed using network pharmacology research methods, which aimed to identify the key active components and potential targets of HGW in treating oligospermia. Experimental validation was finally conducted in animal model. The male-specific pathogen-free Kunming mice were divided into five groups: Sham group, Model group, and HGW groups (8, 16, and 32 g/kg of HGW by gavage for 35 days). Chemical profile and network pharmacology results revealed that potential bioactive compounds were dihydrocinnacasside, isomucronulatol, and 6-gingerol, and the mechanism of which was enriched in regulating spermatogenic stem cells (SSCs), endocrine function, and apoptosis. The administration of HGW significantly improved oligospermia in mice. HGW significantly upregulated the expression of marker proteins in SSCs and the potential targets within the testis simultaneously. Our data indicates that HGW enhances the proliferation of SSCs, and HGW can be a promising therapeutic candidate for oligospermia.

Comprehensive Evaluation of Quality and Differences in Silene viscidula Franch from Different Origins Based on UPLC-ZENO-Q-TOF-MS/MS Compounds Analysis and Antioxidant Capacity

Silene viscidula Franch is mainly produced in southwest China. The region has a vast area and rich climate, which has an impact on the quality of the plants due to the differences in distribution between the origins. There is a lack of systematic research on its chemical compounds in the existing literature, and fewer studies have been reported for the active compounds of this plant. Therefore, high-resolution liquid mass spectrometry was used in this study. Sixty batches of Silene viscidula Franch samples from twenty origins in three provinces were analyzed for compounds. A database of chemical compounds of Silene viscidula Franch was established through node-to-node information in the GNPS molecular network, as well as literature records. The ion fragmentation information obtained was compared with the literature data and analyzed and identified by importing the mass spectrometry software PeakView 1.2. Then, the MarkerView t-test was applied to analyze and identify the compounds of Silene viscidula Franch from different origins. Afterwards, the antioxidant activity of Silene viscidula Franch from different origins was preliminarily evaluated using DPPH and ABTS free radical scavenging assays. The results showed a total of 78 compounds, including 34 steroids, 14 triterpenoid saponins, 30 flavonoid glycosides, and other classes of compounds, such as alkaloids. The cleavage patterns of steroids, triterpenoid saponins, and flavonoids in positive-ion mode were also summarized. Based on the p-value of the t-test (p &lt; 0.05), 29 differential compounds were screened out. The relative contents of saponins and steroidal compounds in these samples were found to be associated with antioxidant activity. This study provided a preliminary reference for the establishment of a comprehensive evaluation system for the quality of Silene viscidula Franch.

ToF-SIMS Investigation of Environmental Effects on Analyte Migration in Matrix Coatings for Mass Spectrometry Imaging Using a Newly Developed Vapor Deposition System.

High resolution mass spectrometry images are of increasing importance in biological applications, such as the study of tissues and single cells. Two promising techniques for this are matrix-enhanced secondary ion mass spectrometry (ME-SIMS) and matrix-assisted laser desorption/ionization (MALDI). For both techniques, the sample of interest must be coated with a matrix prior to analysis, and analytes must migrate into the matrix. The mechanisms involved in this migration and the factors that influence the migration are poorly understood, which lead to difficulties with reproducibility. In this work, a sublimation matrix coater with an effusion cell and sample cooling was developed and built in-house for controlled physical vapor deposition. In this system, sample transfer between the coater and mass spectrometer is possible without breaking vacuum, which facilitates the study of environmental influences on analyte migration. The influence of exposure to ambient air on the migration of two analytes (a lipid and a peptide), which were coated with the matrix α-cyano-4-hydroxycinnamic acid (CHCA), was studied using 3D-SIMS imaging. Although the distribution of analyte in the matrix changed very little after 21 h of storage in vacuum, significant redistribution of the analyte was observed after exposure to ambient air. The magnitude of the effect was greater for the lipid than for the peptide. Further work is needed to determine the role of humidity in the redistribution process and the impact of analyte redistribution on MALDI measurements.

Preparation of Dysprosium(III)-Metal Organic Framework Nanofiber for Exosome Capture and Biomarker Discovery toward Liver Disease.

As an emerging source for liquid biopsy, exosomes hold significant promise for clinical diagnosis. However, commonly used exosome isolation methods (e.g., ultracentrifugation) suffer from low throughput for a large number of clinical samples. Herein, a dysprosium-metal organic framework was synthesized and doped with nanofibers by electrospinning for efficient capture of exosomes from body fluid. With the integration of multichannel of pipet or robot automatic workstation, high throughput exosome isolation can be achieved with clinical samples with high reproducibility. To evaluate the clinical value of the developed method, urinary exosomes were enriched from 34 liver disease samples of different stages for the profiling of metabolites by mass spectrometry. The results showed that HCC, cirrhosis, and healthy controls can be significantly differentiated by the Random Forest classification model. The dysprosium-metal organic framework has promising applications in exosome-based liquid biopsy for large-scale clinical disease diagnosis.

Dysregulation of protein SUMOylation networks in Huntington's disease R6/2 mouse striatum.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation is a post-translational modification with broad cellular effects including via modification of synaptic proteins. Here, we used an optimised SUMO protein-enrichment and mass spectrometry method to identify the protein SUMOylation/SUMO interaction proteome in the context of HD using R6/2 transgenic and non-transgenic (NT) mice. Significant changes in enrichment of SUMOylated and SUMO-interacting proteins were observed, including those involved in presynaptic function, cytomatrix at the active zone scaffolding, cytoskeleton organization, and glutamatergic signaling. Mitochondrial and RNA-binding proteins also showed altered enrichment. Modified SUMO-associated pathways in HD tissue include clathrin-mediated endocytosis signaling, synaptogenesis signaling, synaptic long-term potentiation, and SNARE signaling. To evaluate how modulation of SUMOylation might influence functional measures of neuronal activity in HD cells in vitro, we utilised primary neuronal cultures from R6/2 and NT mice. A receptor internalization assay for the metabotropic glutamate receptor 7 (mGLUR7), a SUMO enriched protein in the mass spec, showed decreased internalization in R6/2 neurons compared to NT. siRNA-mediated knockdown of the E3 SUMO ligase Protein Inhibitor of Activated STAT1 (Pias1), which can SUMO modify mGLUR7, prevented this HD phenotype. In addition, microelectrode array analysis of primary neuronal cultures indicated early hyperactivity in HD cells, while later timepoints demonstrated deficits in several measurements of neuronal activity within cortical neurons. HD phenotypes were rescued at selected timepoints following knockdown of Pias1. Collectively, our results provide a mouse brain SUMOome resource and show that significant alterations occur within the post-translational landscape of SUMO-protein interactions of synaptic proteins in HD mice, suggesting that targeting of synaptic SUMO networks may provide a proteostatic systems-based therapeutic approach for HD and other neurological. Disorders.

The Effect of Biochar Particle Size on the Leaching of Organic Molecules and Macro- and Microelements

Biochar is a carbon-rich material that has recently received attention due to its increasing agronomical potential. The agricultural utilization of biochar relates to its potential to act in the soil as a soil conditioner; nevertheless, complex information on the direct dependence of biochar’s physical properties (texture, particle size) and corresponding leaching and availability of organic molecules (e.g., the polycyclic and heterocyclic organic compounds) and inorganic mineral salts (based on micro- and macroelements) is still inconsistent. Multi-elemental analysis by using inductively coupled plasma atomic emission spectroscopy (ICP-OES) was used to assess the information on the contents and availability of macro- and microelements in studied commercial biochar samples. The results showed a statistically significant indirect relation between an increase in the size fraction of biochar and the content of aqueous-extractable K and Na and the direct relation with the aqueous-extractable Ca, Mg, and P. Compared to the macroelements, the detected contents of aqueous-extractable microelements were almost three orders lower, and the dependence on fraction size was not consistent or statistically significant. In addition, gas chromatography (GC) coupled with mass spectroscopy (MS) was further used to reveal the concentrations of available polycyclic aromatic and heterocyclic compounds in biochar samples. The detected concentrations of these types of organic compounds were far below the certified limits, and a statistically significant indirect correlation with particle size was also observed for all the studied biochar samples. The proposed methodological concept could provide the necessary insights into the description of biochar mineral content and its connection to biochar texture, the physicochemical properties, and the potential of biochar to release nutrients into the soil. These findings could help in the further assessment of biochar as a soil conditioner in modern agriculture.

Coupling Online Size Exclusion Chromatography with Charge Detection-Mass Spectrometry Using Hadamard Transform Multiplexing.

Charge detection mass spectrometry (CD-MS) is a powerful technique for the analysis of large, heterogeneous biomolecules. By directly measuring the charge states of individual ions, CD-MS can measure the masses from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. However, CD-MS is inherently slow because hundreds or thousands of spectra need to be collected to produce adequate ion statistics. The slower speed of CD-MS complicates efforts to couple it with online separation techniques, which limit the number of spectra that can be acquired during a chromatographic peak. Here, we present the application of Hadamard transform multiplexing to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS, with a goal of using SEC for separating complex mixtures prior to CD-MS analysis. We developed a microcontroller to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. Data showed a series of peaks spaced according to the pseudorandom injection sequence, which were demultiplexed with a Hadamard transform algorithm. The demultiplexed data revealed improved CD-MS signals while preserving retention time information. This multiplexing approach provides a general solution to the inherent incompatibilities of online separations and CD-MS detection that will enable a range of applications.

Novel Antioxidant Peptides from Fermented Whey Protein by Lactobacillus rhamnosus B2-1: Separation and Identification by in Vitro and in Silico Approaches.

Whey is a byproduct of the dairy industry and is rich in protein. To enhance the significance of such byproducts and find efficacious antioxidants for combating oxidative stress, this study reported on the preparation, purification, and identification of novel peptides with antioxidant activities from whey protein metabolites following fermentation by Lactobacillus rhamnosus B2-1. The isolation and identification processes involved macroporous adsorption resin column chromatography, gel filtration column chromatography, and liquid chromatography-tandem mass spectrometry. Therein, three novel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were selected to be synthesized, and they demonstrated remarkable antioxidant activities in vitro chemical assays. PKYPVEPF, LEASPEVI, and YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective impact on HepG2 cells under oxidative stress induced by H2O2, increasing the cell viability from 49.02 ± 3.05% to 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%, respectively. Moreover, the peptides boosted the activities of catalase and superoxide dismutase in damaged cells and reduced reactive oxygen species levels. The molecular docking studies highlighted that these antioxidant peptides efficiently bound to key amino acids in the Kelch domain of Keap1, thereby preventing the interaction between Keap1 and Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated substantial antioxidant activity, suggesting their potential for widespread application as functional food additives and ingredients.

Impact of Acupuncture on Human Metabolomic Profiles: A Systematic Review

Background/Objectives: Metabolomics provides insights into the biological underpinnings of disease development and treatment. This systematic review investigated the impact of acupuncture on metabolite levels and associated metabolic pathways using a metabolomic approach. Methods: Five databases (i.e., PubMed, Embase, Scopus, CINAHL, and Cochrane Central) were searched using terms such as “acupuncture” and “metabolites” to retrieve relevant journal articles published through January 2024. Studies utilizing mass spectrometry or nuclear magnetic resonance were included. Risk of bias was evaluated using the Cochrane Risk of Bias tool and the Newcastle–Ottawa scale. Metabolic pathway analysis was conducted using MetaboAnalyst 6.0 to identify common significant pathways affected by acupuncture. Additionally, subgroup pathway enrichment analysis identified metabolites significantly altered in more than two studies. Results: Among 4019 articles, 22 studies met inclusion criteria, examining changes in metabolomic biomarkers before and after acupuncture for various diseases and symptoms. A total of 226 metabolites showed significant changes, with 14 common metabolites altered in more than two studies (glutamine, androsterone glucuronide, choline, citric acid, decanoylcarnitine, estrone, glutathione, glycine, hypoxanthine, lactic acid, pyruvic acid, serine, proline, and sn-glycero-3-phosphocholine). Common pathways affected by acupuncture were glycine, serine, and threonine metabolism, glutathione metabolism, arginine biosynthesis, and glyoxylate and dicarboxylate metabolism. Conclusions: This review provides insights of the metabolomic mechanisms underlying acupuncture, highlighting its impact on specific metabolic pathways. Recognizing these changes can enhance acupuncture’s effectiveness and support the development of personalized treatments. The findings underscore metabolomics as a valuable tool for understanding and optimizing acupuncture for various diseases and symptoms.

Long-Term Photochemical and Microbial Alterations Lead to the Compositional Convergence of Algal and Terrestrial Dissolved Organic Matter.

Lakes are expected to become more active in processing dissolved organic matter (DOM), but the fate of DOM with different origins remains poorly constrained. We conducted long-term incubation experiments (∼1 year) with sole light, sole microbial, and combined light and microbial treatments using DOM from algal and terrestrial sources (DOMa and DOMt, respectively). Fourier transform ion cyclotron resonance mass spectrometry and 16s rRNA were used to analyze the DOM molecular composition and bacterial community, respectively. We observed that DOMa and DOMt converged toward a similar composition under the combined light and microbial treatment, driven by the removal of source-specific compositions along with the production of refractory, carboxylic-rich alicyclic molecules (CRAM). For CRAM enrichment, microbial processes played a greater role for DOMa, while phototransformation was more important for DOMt. The co-occurrence patterns between DOM molecules and bacteria showed that DOM molecular composition influenced the bacterial community. More complex DOM-bacteria interactions were observed for DOMt compared to DOMa, suggesting that greater bacterial cooperation was necessary for transforming DOMt. Collectively, these findings offer new insights into the mechanisms underlying the uniformity of DOM from various sources through prolonged environmental transformations in lakes.

Alterations in the Renin-Angiotensin System in Experimental Septic Shock

OBJECTIVES: To analyze dynamic changes in the renin-angiotensin system (RAS) during septic shock, focusing on angiotensin-converting enzyme (ACE) activity and the balance between angiotensin peptides, using a mass spectrometry method. DESIGN: Experimental septic shock model induced by peritonitis in swine. SETTING: Experimental Laboratory, Department of Intensive Care, Erasme Hospital, Université Libre de Bruxelles. SUBJECTS: Forty time points from eight mechanically ventilated pigs. INTERVENTIONS: Septic shock was induced using intraperitoneal instillation of autologous feces, followed by standardized fluid resuscitation, norepinephrine infusion, antibiotic administration, and peritoneal lavage. MEASUREMENTS AND MAIN RESULTS: The induction of sepsis resulted in a significant increase in plasma renin activity and levels of angiotensin I and II, with a significant decrease in ACE activity observed from 4 hours post-resuscitation and a notable rise in the angiotensin I/angiotensin II ratio at 12 hours. Additionally, a shift toward the angiotensin-(1–7) axis was observed, evidenced by an increased angiotensin-(1–7)/angiotensin II ratio. CONCLUSIONS: The study highlighted dynamic shifts in the RAS during septic shock, characterized by reduced circulating ACE activity, elevated angiotensin I/II ratio, and a shift toward the angiotensin-(1–7) axis. These findings suggest an adaptive response within the RAS, potentially offering new insights into sepsis management and therapeutic targets.