High-resolution Multiple Reaction Monitoring Research Articles

Sample-Specific MS/MS Methods in High-Throughput Mass Spectrometry.

The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.

Comprehensive Proteomic Profiling of Aqueous Humor in Idiopathic Uveitis and Vogt-Koyanagi-Harada Syndrome.

Idiopathic uveitis (IU) and Vogt-Koyanagi-Harada (VKH) syndrome are common types of uveitis. However, the exact pathological mechanisms of IU and VKH remain unclear. Proteomic analysis of aqueous humor (AH), the most easily accessible intraocular fluid and a key site of uveitis development, may reveal potential biomarkers and elucidate uveitis pathogenesis. In this study, 44 AH samples, including 12 IU cases, 16 VKH cases, and 16 controls, were subjected to label-free quantitative proteomic analysis. We identified 557 proteins from a comprehensive spectral library of 634 proteins across all samples. The AH proteomic profiles of the IU and VKH groups were different from those of the control group. Differential analysis revealed a shared pattern of extracellular matrix disruption and downregulation of retinal cellular proteins in the IU and VKH groups. Enrichment analysis revealed a protein composition indicative of inflammation in the AH of the IU and VKH groups but not in that of the control group. In the IU and VKH groups, innate immunity played an important role, as indicated by complement cascade activation and overexpression of innate immune cell markers. Extreme gradient boosting (XGBoost), an efficient and robust machine learning algorithm, was subsequently used to screen potential biomarkers for classifying the IU, VKH, and control groups. Transferrin and complement factor B were deemed the most important and represent a promising biomarker panel. These proteins were validated by high-resolution multiple reaction monitoring (HR-MRM) in an independent validation cohort. A classification decision tree was subsequently built for the diagnosis. Our findings further the understanding of the underlying molecular mechanisms in IU and VKH and facilitate the development of potential therapeutic and diagnostic strategies.

Discrimination and prediction of Qingzhuan tea storage year using quantitative chemical profile combined with multivariate analysis: Advantages of MRMHR based targeted quantification metabolomics

The duration of storage significantly influences the quality and market value of Qingzhuan tea (QZT). Herein, a high-resolution multiple reaction monitoring (MRMHR) quantitative method for markers of QZT storage year was developed. Quantitative data alongside multivariate analysis were employed to discriminate and predict the storage year of QZT. Furthermore, the content of the main biochemical ingredients, catechins and alkaloids, and free amino acids (FAA) were assessed for this purpose. The results show that targeted marker–based models exhibited superior discrimination and prediction performance among four datasets. The R2Xcum, R2Ycum and Q2cum of orthogonal projection to latent structure–discriminant analysis discrimination model were close to 1. The correlation coefficient (R2) and the root mean square error of prediction of the QZT storage year prediction model were 0.9906 and 0.63, respectively. This study provides valuable insights into tea storage quality and highlights the potential application of targeted markers in food quality evaluation.

Evaluation of different QuEChERS-based methods for the extraction of 48 wastewater-derived organic contaminants from soil and lettuce root using high-resolution LC-QTOF with MRMHR and SWATH acquisition modes

The reuse of treated wastewater in agriculture is an important route of introducing a large number of organic contaminants into the agroecosystem. In this study, a modified QuEChERS-based approach was developed for rapid, simple, and simultaneous extraction of 48 organic wastewater-derived contaminants from soil and lettuce root. Twenty-two different (modification) scenarios of the known (or original) QuEChERS method have been tested, in order to obtain best and well-compromised recoveries for all target compounds for soil and roots. Finally, a common method was chosen for both matrices consisting of a single extraction step using EDTA-Mcllvaine buffer and the unbuffered Original QuEChERS salts. Method performance was accomplished by liquid chromatography coupled with high-resolution mass spectrometry on a QToF-MS system using two different acquisition modes, the ultra-fast high-resolution multiple reaction monitoring (MRMHR) mode and the innovative Sequential Window Acquisition of All Theoretical Fragment-Ion (SWATH) mode. Performance characterization was evaluated in terms of recovery, linearity, intra-day precision, method detection limits (MDLs), method quantification limits (MQLs), and matrix effect (ME). Recoveries in MRMHR mode ranged from 63 to 111% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in MRMHR mode and from 56 to 121% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in SWATH. Whereas, MQLs ranged from 0.03 to 0.92 ng g−1 in MRMHR and from 0.03 to 82 ng g−1 in SWATH for lettuce root, and from 0.02 to 0.44 ng g−1 in MRMHR and 0.02 to 0.14 ng g−1 in SWATH for soil. The method was then applied to follow the target compounds in soil and lettuce root, where the system lettuce-soil was irrigated with treated wastewater under real greenhouse conditions. Five and 17 compounds were detected in lettuce root and soil, respectively.Graphical

Proteomic analysis of human aqueous humor from fuchs uveitis syndrome

Fuchs uveitis syndrome (FUS) is a commonly misdiagnosed uveitis syndrome often presenting as an asymptomatic mild inflammatory condition until complications arise. The diagnosis of this disease remains clinical because of the lack of specific laboratory tests. The aqueous humor (AH) is a complex fluid containing nutrients and metabolic wastes from the eye. Changes in the AH protein provide important information for diagnosing intraocular diseases. This study aimed to analyze the proteomic profile of AH in individuals diagnosed with FUS and to identify potential biomarkers of the disease. We used liquid chromatography-tandem mass spectrometry-based proteomic methods to evaluate the AH protein profiles of all 37 samples, comprising 15 patients with FUS, six patients with Posner-Schlossman syndrome (PSS), and 16 patients with age-related cataract. A total of 538 proteins were identified from a comprehensive spectral library of 634 proteins. Subsequent differential expression analysis, enrichment analysis, and construction of key sub-networks revealed that the inflammatory response, complement activation and hypoxia might be crucial in mediating the process of FUS. The hypoxia inducible factor-1 may serve as a key regulator and therapeutic target. Additionally, the innate and adaptive immune responses are considered dominant in the patients with FUS. A diagnostic model was constructed using machine-learning algorithm to classify FUS, PSS, and normal controls. Two proteins, complement C1q subcomponent subunit B and secretogranin-1, were found to have the highest scores by the Extreme Gradient Boosting, suggesting their potential utility as a biomarker panel. Furthermore, these two proteins as biomarkers were validated in a cohort of 18 patients using high resolution multiple reaction monitoring assays. Therefore, this study contributes to advancing of the current knowledge of FUS pathogenesis and promotes the development of effective diagnostic strategies.

Myopia Control Efficacy and Long-Term Safety of a Novel Orthokeratology Lens (MESOK Study)-A Randomized Controlled Clinical Trial Combining Clinical and Tear Proteomics Data.

Myopia control efficacy and long-term safety of the Breath-O-Correct orthokeratology (OK) lens was evaluated in a 2-year randomized, single vision (SV) spectacle lens-controlled, single-blind clinical trial combining clinical and tear proteomics data. A total of 71 children (43 OK, 9.8 ± 1.3 years; 28 SV, 9.5 ± 1.4 years) completed the 2-year study. Axial length (AL), cycloplegic refraction, clinical safety parameters (best-corrected visual acuity, central cornea thickness, corneal endothelial health, ocular surface disease index), and quantitative tear proteomics were evaluated by masked examiners. Mean 2-year-normalized AL elongations in the OK and SV groups differed significantly (p = 0.03) and were 0.37 ± 0.37 mm and 0.60 ± 0.41 mm, respectively. OK-mediated myopia control efficacy was 37.1%. No significant difference was found in clinical safety parameters of both groups (p > 0.10), except for a thinner central corneal thickness in the OK group (p = 0.01). Proteomics revealed modest OK lens-mediated effects on immune response proteins, including an increased abundance of haptoglobin at 6 and 12 months and a decreased abundance of two proteins (neutrophil defensin 3 and histone 4) at 6 months. The changes were further validated using a high-resolution multiple-reaction monitoring (MRMHR) mass spectrometry. In summary, the Breath-O-Correct OK lens significantly reduced AL elongation in schoolchildren without adverse clinical effects or subclinical inflammatory responses.

Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring

Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS2 fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRMHR). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS2 fragmentation mode based on the measurement of 13C- or 15N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.

Localization of Multiple O-Linked Glycans Exhibited in Isomeric Glycopeptides by Hot Electron Capture Dissociation.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various O-glycoforms in isomeric glycopeptides using hot electron capture dissociation (hot ECD) in liquid chromatography-tandem mass spectrometry. To capture low abundant glycosylated species, a prototype of a ZenoTOF 7600 system incorporating an efficient electron-activated dissociation device to perform hot ECD was operated in targeted or scheduled high-resolution multiple reaction monitoring workflows. In addition, Zeno trap pulsing was activated to enhance the sensitivity of the time-of-flight mass spectrometer. Sixty-nine O-glycopeptides of the long O-glycopeptides in tryptic bovine fetuin digest were obtained with a relative abundance range from 100 to 0.2%, which included sialylated glycans with Neu5Ac and Neu5Gc.

Persistence, decontamination and dietary risk assessment of propyrisulfuron residue in natural paddy field environment using QuEChERS@UPLC-Q-TOF-MS/MS

A modified QuEChERS technology combined with UPLC-Q-TOF-MS/MS residue analytical method for propyrisulfuron in natural paddy field environment was developed. Propyrisulfuron residues in paddy water, soil, rice plants, brown rice and rice husk samples was extracted with 1.0 % acetic acid-acetonitrile (v/v) solution and separated on a reversed-phase C18 column with a mobile phase of 0.1 % formic acid water-acetonitrile eluted by isometric elution (60/40, v/v), and quantified in high-resolution multiple reaction monitoring (MRMHR) scanning mode providing more accurate confirmatory information. The lower limit of quantification (LOQ) of propyrisulfuron in all the five paddy matrices was in a range of 1.24 ∼ 3.85 µg/kg, and all calibration curves showed good linearity (R2 > 0.991) in a certain concentration range (10 ∼ 1000 µg/kg). At the spiked level of 10 ∼ 200 µg/kg, the intra-day and inter-day average recoveries and relative standard deviation were 82.80 ∼ 106.93 % and 1.81 ∼ 5.51 %, 83.05 ∼ 105.84 % and 2.25 ∼ 4.61 %, respectively, for analysis on three consecutive days, and there was a good retention time repeatability of 15 consecutive injections with RSD of 0.15 ∼ 0.17 %, indicating a high sensitivity, specificity, accuracy and precision. All paddy filed environmental samples frozen at −20 °C were analyzed within 10 weeks after collection for a good storage stability. Propyrisulfuron dissipated in paddy water, soil and rice plants at application doses of 30, 45 and 60 g a.i./ha with half-lives of 1.37 ∼ 1.81, 4.10 ∼ 5.35 and 3.70 ∼ 4.42 days in 2020, 1.44 ∼ 2.04, 4.27 ∼ 5.83 and 3.68 ∼ 4.49 days in 2021, respectively. The final propyrisulfuron residual levels of two years in brown rice, rice husks and rice plants samples prior to harvest were lower than 8.18 µg/kg, and its removal rate was in a range of 1.40 to 98.27 % by several simple available household technologies. Moreover, the chronic dietary risk in brown rice at recommended dose for Chinese residents of different age groups was low and acceptable for daily consumption.

Fast screening and confirmation of 73 common prohibited compounds in cosmetics by ultra performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间高分辨质谱(UPLC-Q-TOF HRMS)同时快速筛查确证化妆品中73种常见禁用物质的方法。样品经饱和氯化钠溶液分散均匀后,采用含0.2%甲酸的乙腈溶液超声提取,50 mg PSA净化,以8000 r/min高速冷冻离心除脂,采用Waters Acquity HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)分离。采用多反应监测高分辨扫描模式(MRM HR),以保留时间、一级母离子精确质量数、同位素丰度比和二级子离子精确质量数实现化妆品中73种禁用物质的快速筛查和确证,基质匹配外标法定量。实验比较了不同提取溶剂、净化吸附剂、色谱条件和质谱扫描模式对73种禁用物质测定的影响,并考察了膏霜剂和水剂的基质效应。结果表明,73种禁用物质线性关系良好,相关系数(R2)>0.99;检出限为5~150 μg/kg;定量限为15~450 μg/kg;膏霜剂及水剂两种基质在3个加标水平下的回收率为60.3%~130.3%,日内、日间RSD分别为0.8%~10.0%(n=6)和1.1%~15.0%(n=3)。日常风险监测中检出磺胺甲基异噁唑、甲基泼尼松、林可霉素、对乙酰氨基酚、甲氧苄啶、阿法骨化醇、倍他米松戊酸酯、溴莫尼定、氯霉素、氯苯那敏、氯倍他索丙酸酯、克罗米通、益康唑、酮康唑、泼尼松醋酸酯和泼尼松,检出含量范围为0.5~1136.1 mg/kg。该方法准确、快速、简便,可用于化妆品中73种常见禁用物质的检测。

Validation of quick and highly specific quantitation method of mycotoxin in cocoa beans by high resolution multiple reaction monitoring technique for reference materials analysis

This article discussed a development, optimization, and validation of modified QuEChERS method with liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) operated in time-of-flight multiple-reaction-monitoring (TOF-MRM) mode for the analysis of ochratoxin A and aflatoxins (B1, B2, G1, G2) in cocoa beans. The recoveries for the optimized and validated method at 2 and 5 ng/g were in the range of 75.4–107.8 % with RSDs below 11.5 %. Good sensitivity was obtained for all mycotoxins with method limit of quantification oranged between 0.7 to 1.4 ng/g. New identification criteria was proposed using multiple reaction monitoring (MRM) transition product ions mass accuracy to prevent both false positive and negative. Finally, the proposed method was used to analyze mycotoxin residues in artificially contaminated cocoa beans samples for reference material production.

Quantitative Proteomic Analysis for High- and Low-Aflatoxin-Yield Aspergillus flavus Strains Isolated From Natural Environments.

The molecular mechanisms underlying aflatoxin production have been well-studied in strains of the fungus Aspergillus flavus (A. flavus) under artificial conditions. However, aflatoxin biosynthesis has rarely been studied in A. flavus strains isolated from field conditions with different aflatoxin-producing ability. In the present study, tandem mass tag (TMT) labeling and high-performance liquid chromatography (HPLC) coupled with tandem-mass spectrometry analysis were used for proteomic quantification in natural isolates of high- and low-aflatoxin-yield A. flavus strains. Additionally, findings obtained using the TMT-labeling method were validated using the high-resolution multiple reaction monitoring (MRM-HR) method. In total, 4,363 proteins were quantified, among which 1,045 proteins were differentially expressed between the high- and low-aflatoxin-yield A. flavus strains. Bioinformatics analysis showed that the up-regulated proteins were significantly enriched in carbon-related metabolism and the biosynthesis of secondary metabolites, whereas the down-regulated proteins were enriched in oxidative phosphorylation. Moreover, GST proteins were found to be significantly down-regulated in high-yield A. flavus strains; this result contradicted previous findings obtained from A. flavus strains grown under artificial conditions. In summary, our study provides novel insights into aflatoxin regulation in A. flavus under field conditions and could facilitate the development of various strategies for the effective control of aflatoxin contamination in food crops.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

Determination of Pesticides in Apples by High-Performance Liquid Chromatography–Mass Spectrometry (HPLC–MS) with High-Resolution Multiple Reaction Monitoring

High-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF–MS) is reported for quantitative determination of pesticide residues in apples. After a simple, rapid extraction, 300 mg MgSO4, 50 mg primary secondary amine (PSA), and 250 mg octadecyl (C18) were used for sample clean-up to ensure efficient recovery. The pesticides were quantified by HPLC/QTOF–MS using high-resolution multiple reaction monitoring (HR-MRM). Matrix-matched external standard calibration was used for quantitative analysis. The developed method was validated by spiking blank samples with the analytes and provided satisfactory accuracy verified by recoveries from 78.9% to 109.4% and good precision (relative standard deviation ≤12.4%). The limits of quantification for all analytes were between 0.85 and 3.0 µg/kg. The method was successfully applied to the analysis of forty batches of real samples, demonstrating its effectiveness for routine application.

Comparison of high resolution mrm and sequential window acquisition of all theoretical fragment-ion acquisition modes for the quantitation of 48 wastewater-borne pollutants in lettuce

Screening of a large number of chemicals of emerging concern is highly desirable for the control of crops irrigated with reclaimed water since it is considered an alternative water source of great value. This study describes a high resolution mass spectrometry approach for developing methods for quantification in lettuce leaves of 48 different wastewater-borne pollutants (including analgesics and anti-inflammatories, anti-hypertensives, antifungal agents, lipid regulators, psychiatric drugs and stimulants, β-blockers, antibiotics, antimycotics, and sweeteners) frequently found in water resources. In this respect, a simple and fast QuEChERS-based method for the determination of contaminants in lettuce has been developed. During extraction, the use of formic acid was adopted to further improve the results of some problematic compounds (e.g., fenofibrate, furosemide, metronidazole, oxcarbazepine, sulfanilamide). High resolution multiple reaction monitoring (MRMHR) and SWATH acquisition were compared in term of accuracy, repeatability, sensitivity, linearity and matrix effect. Both methods provided similar recoveries between 80 and 120% in lettuce leaves, although sulfanilamide, ciprofloxacin, and sulfamethazine presenting values of 26.8, 27.8, and 28.4% in MRMHR and 25, 33.9, and 35% in SWATH, respectively. The effectiveness of a two-step cleanup on analyte recovery was also assessed and matrix effects were also taken into consideration during the method validation. The developed method allows the simultaneous quantitative analysis of 48 compounds (drug residues and metabolites) in lettuce leaves irrigated with treated wastewater for human consumption. Application of the present method to lettuce crops growth in controlled conditions showed the presence of 14 out 48 studied compounds with similar concentrations in both acquisition modes ranging from 3.3 and 1.3 ng g − 1 for climbazole (for MRMHR and SWATH, respectively) to 33.2 and 17.7 ng g − 1 for sulfamethazine. Drug residues such as carbamazepine (6.0 and 8.5 ng g − 1), and its metabolite carbamazepine epoxide (18.1 and 16.5 ng g − 1), frequently found in wastewater effluents, were also detected.

Development and application of a QuEChERS method with liquid chromatography-quadrupole time of flight-mass spectrometry for the determination of 50 wastewater-borne pollutants in earthworms exposed through treated wastewater

Soil-dwelling earthworms are valuable sentinels in soil pollution monitoring and, in case of wastewater reuse for agricultural irrigation, they are continuously exposed to a multitude of organic micro-pollutants. In the present work, an analytical methodology for the determination of 50 wastewater-borne pollutants in earthworms (Lumbricus terrestris) using a fast extraction and sensitive detection method was developed. In total, 17 protocols based on QuEChERS extraction methods were evaluated including the choice of extraction salt (EN vs Original) and the solvent pH as well as the cleanup type. EN with cleanup on Oasis PRiME HLB (P-16) provided the best overall performance. Compound quantification was accomplished by liquid chromatography-mass spectrometry on a QToF-MS system using the ultra-fast high-resolution multiple reaction monitoring (MRMHR) mode. The method quantification limits ranged from 0.002 to 1.6 ng g−1. In comparison to previously reported methods the present protocol afforded improved accuracy with recovery rates exceeding 80%. The validated method was applied to the analysis of 36 earthworm samples originating from laboratory experiments and fields that had been irrigated with treated wastewater. Among the eight analytes detected in field samples, the highest concentration levels were measured for gemfibrozil (13 ng g−1) and caffeine (12 ng g−1). To the best of our knowledge, this is the first report of the transfer of wastewater-borne contaminants to earthworms following irrigation under natural farming practices.

Melatonin down-regulates steroidal hormones, thymocyte apoptosis and inflammatory cytokines in middle-aged T. cruzi infected rats

Chagas disease, triggered by the flagellate protozoan Trypanosoma cruzi (T. cruzi) plays a potentially threat to historically non-endemic areas. Considerable evidence established that the immuno-endocrine balance could deeply influence the experimental T. cruzi progression inside the host's body. A high-resolution multiple reaction monitoring approach (MRMHR) was used to study the influence of melatonin on adrenal and plasma steroidal hormones profile of T. cruzi infected Wistar rats. Young (5 weeks) and middle-aged (18 months) male Wistar rats received melatonin (5 mg/Kg, orally) during the acute Chagas disease. Corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, progesterone and melatonin concentration were evaluated. Interleukin-1 alpha and β (IL-1α and β), IL-6 and transforming growth factor beta (TGF-β) were also analyzed. Our results revealed an increased production of corticosterone, cortisone, cortisol and aldosterone in middle-aged control animals, thus confirming the aging effects on the steroidal hormone profile. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats. Melatonin treatment reduced the corticosterone, 11-DHC, cortisol, cortisone, aldosterone and progesterone in response to T. cruzi infection. Decreased IL-1 α and β concentrations were also found in melatonin treated middle-aged infected animals. Melatonin treated middle-aged control rats displayed reduced concentrations of TGF-β. Melatonin levels were significantly higher in all middle-aged rats treated animals. Reduced percentages of early and late thymocyte apoptosis was found for young and middle-aged melatonin supplemented rats. Finally, our results show a link between the therapeutic and biological effects of melatonin controlling steroidal hormones pathways as well as inflammatory mediators.

Alteration of retinal metabolism and oxidative stress may implicate myopic eye growth: Evidence from discovery and targeted proteomics in an animal model

Myopia, the most common cause of impaired vision, may induce sight- threatening diseases or ocular complications due to axial elongation. The exact mechanisms underlying myopia development have received much attention and understanding of these is necessary for clinical prevention or therapeutics. In this study, quantitative proteomics using Isotope Coded Protein Label (ICPL) was applied to identify differentially regulated proteins in the retinas of myopic chicks and, from their presence, infer the possible pathogenesis of excessive ocular elongation. Newly hatched white leghorn chicks (n = 15) wore -10D and + 10D lenses bilaterally for 3 and 7 days, respectively, to develop progressive lens-induced myopia (LIM) and hyperopia (LIH). Retinal proteins were quantified with nano-liquid chromatography electrospray ionization coupled with tandem mass spectrometry (nanoLC-ESI-MS/MS). Bioinformatics analysis of differentially regulated proteins revealed that the majority originated from the cytoplasmic region and were related to various metabolic, glycolytic, or oxidative processes. The fold changes of four proteins of interest (vimentin, apolipoprotein A1, interphotoreceptor retinoid binding protein, and glutathione S-transferase) were further confirmed by a novel high-resolution multiple reaction monitoring mass spectrometry (MRM-HR) using a label-free approach. SignificanceDiscovery of effective protein biomarkers of myopia has been extensively studied to inhibit myopia progression. This study first applied lens-induced hyperopia and myopia in the same chick to maximize the inter-ocular differences, aiming to discover more protein biomarker candidates. The findings provided new evidence that myopia was metabolism related, accompanied by altered energy generation and oxidative stress at retinal protein levels. The results in the retina were also compared to our previous study in vitreous using ICPL quantitative technology. We have now presented the protein changes in these two adjacent tissues, which may provide extra information of protein changes during ocular growth in myopia.

Proteomic analysis of rat kidney under maleic acid treatment by SWATH-MS technology.

Maleic acid is an industrial-grade chemical that is often used in adhesives, stabilizers, and preservatives. It is unknown whether long-term consumption of maleic acid modified starch is harmful to humans. However, many studies have indicated that maleic acid causes renal tubular damage in animal models, even as the associated pathways remain unclear. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is the most innovative of the label-free quantitative technologies which have better quantification performance. Therefore, SWATH technology was used to investigate the effect of maleic acid on the rat kidney proteome in this study. Sprague-Dawley(SD) rats were treated with 0 mg/kg (control), 6 mg/kg (low-dose), 10 mg/kg (medium-dose), and 60 mg/kg (high-dose) of maleic acid. After kidney protein extraction, 28% SDS-PAGE was used, followed by in-gel digestion and desalting. Next, the samples were analyzed with ultra-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS), and data-dependent acquisition (DDA) and SWATH technology were also used. The gene ontology and pathway analysis were accomplished. Ultimately, these protein biomarkers were validated by using scheduled high-resolution multiple reaction monitoring (sMRMHR ). Comparisons of the control group with the other three groups revealed that 95, 130, and 103 proteins were expressed at significantly different levels in the control group and in the low-dose, medium-dose, and high-dose groups, respectively. According to the gene ontology analysis, the major processes that these proteins were involved in were metabolic processes, biological regulation, cellular processes, and responses to stimuli; the major functions that these proteins were involved in were binding, hydrolase activity, catalytic activity, and oxidoreductase activity; and the major cellular components hat they were involved in were the cytoplasm, extracellular region, membrane, and mitochondria. According to the KEGG pathway analysis, these proteins were involved in 35 pathways, five of which, the carbohydrate metabolism, folate biosynthesis, renal tubular resorption, amino acid metabolism, and Ras signaling pathways, are discussed in this study. Ultimately, 19 proteins involved in 12 important pathways were validated by sMRMHR . It was demonstrated that maleic acid caused insufficient energy production, which might lead to a decrease in the activity of the sodium-potassium ATP pump and hydrogen ion ATP pump, which could in turn have caused renal tubular resorption and hydrogen ion regulation to be blocked, thus leading to the accumulation of hydrogen ions in the renal tubules, which would then result in renal tubular acidification followed finally by Fanconi syndrome.

Development and application of a SPE-LC-QTOF method for the quantification of micropollutants of emerging concern in drinking waters from the Brazilian capital

Abstract The Brazilian Federal District is located under a savannah climate characterized by an intense dry season, resulting in severe problems related to water availability. This situation motivates the search for alternate water sources, leading to the indirect water reuse in the Paranoa Lake, an artificial reservoir located in the heart of the Brazilian capital. This work aimed the assessment of 35 micropollutants of emerging concern by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry. Solid-phase extraction conditions (Oasis HLB) were optimized by a factorial design. Analytes were then assessed in 0.45 μm filtered samples (1 L) under three different pH by matrix-matched calibration with standard additions and high-resolution multiple reaction monitoring acquisition. Caffeine, bisphenol A and the insect repellant DEET were detected in all investigated samples (n = 14), followed by atrazine (86% of the samples), carbamazepine (71%), paraxanthine (71%), mefenamic acid (64%), nicotine (57%), atenolol (43%) and sulfamethoxazole (43%). Although not frequently detected, the reproductive hormones, estrone, 17β-estradiol and estriol, as well as the ovulation inhibitor 17α-ethinylestradiol were detected in a sample from the Paranoa Lake WTP in concentrations ranging from 3.71 ± 0.08 and 5.6 ± 0.1 ng L−1. Under these levels, risk quotients indicate the potential for adverse effects to both human health and the environment.