Exactive Plus Research Articles

Gábor Transform-Based Antibody Quantitation in Serum: An Interlaboratory Liquid Chromatography/High-Resolution Mass Spectrometry Investigation.

Therapeutic monoclonal antibodies (t-mAbs) are crucial for treating various conditions, including cancers and autoimmune disorders. Accurate quantitation and pharmacokinetic monitoring of t-mAbs in serum are essential, but current methods like ligand binding assays (LBAs) and bottom-up peptide liquid chromatography-tandem mass spectrometry (LC-MS/MS) can lack the sensitivity and specificity needed to meet clinical demands. Emerging techniques using high-resolution mass spectrometry (HRMS) in top-down and middle-up approaches offer improved ability to accurately quantify mAb proteoforms apart from degradation products by keeping the sample proteins intact or minimizing digestion. This study describes the first use of Gábor transform (GT)-based iFAMS Quant+ software to quantify a t-mAb (vedolizumab) from ∼400 samples using an Agilent 6545XT AdvanceBio Q-TOF at the University of Oregon. These results are compared to a previously validated laboratory-developed test (LDT) from Mayo Clinic utilizing a Thermo Q Exactive Plus Orbitrap. The Mayo method used conventional extracted ion chromatograms (XICs) of select charge states for quantitation, while the iFAMS Quant+ method utilized GT-based charge state deconvolution, background subtraction, and signal integration. Calibration and quality control (QC) analyses and Passing-Bablok regression of 351 subject samples demonstrated excellent agreement between the two methods. The iFAMS Quant+ workflow exhibited unique advantages for characterizing interferents and analyte signal anomalies due to its deconvolution-based approach.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Bacillus subtilis Induces Human Beta Defensin-2 Through its Lipoproteins in Human Intestinal Epithelial Cells.

Human intestinal epithelial cells (IECs) play an important role in maintaining gut homeostasis by producing antimicrobial peptides (AMPs). Bacillus subtilis, a commensal bacterium, is considered a probiotic. Although its protective effects on intestinal health are widely reported, the key component of B. subtilis responsible for its beneficial effects remains elusive. In this study, we tried to identify the key molecules responsible for B. subtilis-induced AMPs and their molecular mechanisms in a human IEC line, Caco-2. B. subtilis increased human beta defensin (HBD)-2 mRNA expression in a dose- and time-dependent manner. Among the B. subtilis microbe-associated molecular patterns, lipoprotein (LPP) substantially increased the mRNA expression and protein production of HBD-2, whereas lipoteichoic acid and peptidoglycan did not show such effects. Those results were confirmed in primary human IECs. In addition, both LPP recognition and HBD-2 secretion mainly took place on the apical side of fully differentiated and polarized Caco-2 cells through Toll-like receptor 2-mediated JNK/p38MAP kinase/AP-1 and NF-κB pathways. HBD-2 efficiently inhibited the growth of the intestinal pathogens Staphylococcus aureus and Bacillus cereus. Furthermore, LPPs pre-incubated with lipase or proteinase K decreased LPP-induced HBD-2 expression, suggesting that the lipid and protein moieties of LPP are crucial for HBD-2 expression. Q Exactive Plus mass spectrometry identified 35 B. subtilis LPP candidates within the LPP preparation, and most of them were ABC transporters. Taken together, these results suggest that B. subtilis promotes HBD-2 secretion in human IECs mainly with its LPPs, which might enhance the protection from intestinal pathogens.

Pharmacokinetics and metabolite identification of 23-hydroxybetulinic acid in rats by using liquid chromatography-mass spectrometry method

23-hydroxybetulinic acid (23-HA), a main bioactive component isolated from Pulsatilla chinensis (Bunge) Regel, exhibits various pharmacological activities, such as antimelanoma, antileukemia, anti-colon cancer, and antihepatotoxicity. Although the main active ingredient anemoside B4 (AB4) from this plant has been well studied, research on its active metabolite 23-HA is limited. In the present study, a validated HPLC-QQQ-MS/MS method was established for the quantification of 23-HA in rat plasma. Pharmacokinetics analysis showed that the absorption and elimination of 23-HA in rats were rapid, with an oral bioavailability as 12.9 %. After oral administration with 50 mg/kg 23-HA for SD rats, the plasma, urine, feces, and bile samples were collected and analyzed by UPLC-Q Exactive Plus MS and HPLC-QQQ-MS/MS. Seventeen metabolites of 23-HA were identified, and its major metabolic pathways included oxidation, hydration, sulfation, and glucuronidation. This study highlights the first detailed investigation of 23-HA’s pharmacokinetics in rats along with its metabolism in vivo, and will provide robust evidence for further research and clinical application of 23-HA.

Efficient rhodamine B dye degradation by red mud-grapefruit peel biochar catalysts activated persulfate in water.

The continuous and rapid development of textile industry intensifies rhodamine B dye (RhB) wastewater pollution. Meanwhile, massive red mud (RM) solid waste generated by the industrial alumina production process poses detrimental effects to the environment after leaching. For resource utilization and to reduce the expansion of RhB pollution, RM and peel red mud-biochar composite (RMBC) catalyst were synthesized in activating peroxydisulfate (PDS) for RhB degradation. Firstly, characterization results showed that compared to RM, RMBC had a higher content of catalytically active metals (Fe, Al, Ti) (higher than 0.92-4.18%), smaller pore size, and larger specific surface area (10 times), which verified RMBC had more potential catalytic oxidation activity. Secondly, under optimal dosage (catalyst, PDS), pH 4.6, and 20 mg L-1 RhB, it was found that the RhB degradation ratio of RM was 76.70%, which was reduced to 41% after three cycles, while that of RMBC was 89.98% and 67%, respectively. The results indicated that the performance of RMBC was significantly superior to that of RM. Furthermore, the quenching experiments, electron paramagnetic resonance spectroscopy tests, FTIR, and XPS analysis showed the function of O-H, C=O, C-O, Fe-O, and Fe-OH functional groups, which converted the PDS to the active state and hydrolyzed it to produce free radicals ([Formula: see text], 1O2, [Formula: see text]) for RhB degradation. And, Q Exactive Plus MS test obtained that RhB was degraded to CO2, H2O, and intermediate products. This study aimed to raise a new insight to the resource utilization of RM and the control of dye pollution.

Effects of processing on component interactions and peptide profiles of preterm infant formula based on peptidomics by Q Exactive plus analysis

The study aimed to investigate the effects of processing on the component interactions, the peptide profile as well as the bioactive peptides of preterm infant formula after digestion through EASY-nLC1200 Q Exactive Plus system. Results indicated that whey proteins and caseins transferred from serum and pellet fractions to fat fraction to recombine the milk fat globule membrane destroyed after homogenization. Protein interactions caused the changes of nitrogen distribution, protein profiles, and morphology after pasteurization and spray drying. Peptidomics analysis demonstrated that the component interactions of preterm infant formula affected the peptide profiles after digestion shown by the different peptide intensities of certain amino acid compositions and the totally different abundance of the peptides in heatmap. The hydrolysates of five groups exhibited 334, 274, 421, 214, and 265 unique peptides belonging to 144, 116, 185, 101, and 231 master accession proteins respectively. The top 8 abundant proteins identified were β-casein, Lipocln, αS1-casein, αS2-casein, κ-casein, bovine Glycosylation-dependent cell adhesion molecule, α-lactalbumin, and Albumin. The number of bioactive peptides increased after processing but were different from each other. In total, component interactions during processing of preterm infant formula could change the peptide profile and bioactive peptides after in vitro digestion.

Non-targeted analysis and risk assessment of intentionally and non-intentionally added substances migrating from the emerging biodegradable food contact material poly(butylene adipate-co-terephthalate)/modified starch blend film

Emerging biodegradable food contact materials (FCMs) are omnipresent in food handling and processing. However, similar to conventional FCMs, small molecules can migrate into the food matrix during contact. Extensive evaluation of the migration of chemicals, added intentionally and non-intentionally, from FCMs into food is a crucial step in the safety assessment of such materials. In this study, non-targeted screening was first performed to analyze migrants from poly(butylene adipate-co-terephthalate)/modified starch (PBAT/MFS) FCM using gas chromatography–mass spectrometry and ultra-performance liquid chromatography-Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Sixteen volatile migrants were identified, and 134 non-volatile compounds were screened for dissolution precipitates and food simulants using a self-built and commercially-available databases. The risk ranking for migrants revealed that 13 small molecules had high scores and were assigned as Risk I substances of great concern. Among them, pesticide residues and mycotoxins non-intentionally introduced from MFS blending are of great concern.

Application of infrared matrix-assisted laser desorption electrospray ionization mass spectrometry for morphine imaging in brain tissue

Here, we present a method developed for the analysis of spatial distributions of morphine in mouse brain tissue using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer. The method is also capable of evaluating spatial distributions of the antiretroviral drug abacavir. To maximize sensitivity to morphine, we analyze various Orbitrap mass spectrometry acquisition modes utilizing signal abundance and frequency of detection as evaluation criteria. We demonstrate detection of morphine in mouse brain and establish that the selected ion monitoring mode provides 2.5 times higher sensitivity than the full-scan mode. We find that distributions of morphine and abacavir are highly correlated with the Pearson correlation coefficient R = 0.87. Calibration showed that instrument response is linear up to 40 pg/mm2 (3.8 μg/g of tissue).Graphical abstract

Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Plasma lipidome and risk of atrial fibrillation: results from the PREDIMED trial

The potential role of the lipidome in atrial fibrillation (AF) development is still widely unknown. We aimed to assess the association between lipidome profiles of the Prevención con Dieta Mediterránea (PREDIMED) trial participants and incidence of AF. We conducted a nested case–control study (512 incident centrally adjudicated AF cases and 735 controls matched by age, sex, and center). Baseline plasma lipids were profiled using a Nexera X2 U-HPLC system coupled to an Exactive Plus orbitrap mass spectrometer. We estimated the association between 216 individual lipids and AF using multivariable conditional logistic regression and adjusted the p values for multiple testing. We also examined the joint association of lipid clusters with AF incidence. Hitherto, we estimated the lipidomics network, used machine learning to select important network-clusters and AF-predictive lipid patterns, and summarized the joint association of these lipid patterns weighted scores. Finally, we addressed the possible interaction by the randomized dietary intervention.Forty-one individual lipids were associated with AF at the nominal level (p < 0.05), but no longer after adjustment for multiple-testing. However, the network-based score identified with a robust data-driven lipid network showed a multivariable-adjusted ORper+1SD of 1.32 (95% confidence interval: 1.16–1.51; p < 0.001). The score included PC plasmalogens and PE plasmalogens, palmitoyl-EA, cholesterol, CE 16:0, PC 36:4;O, and TG 53:3. No interaction with the dietary intervention was found. A multilipid score, primarily made up of plasmalogens, was associated with an increased risk of AF. Future studies are needed to get further insights into the lipidome role on AF.Current Controlled Trials number, ISRCTN35739639.

Offline Peptide Fractionation and Parallel Reaction Monitoring MS for the Quantitation of Low-Abundance Plasma Proteins.

Mass spectrometry (MS)-based protein quantitation is an attractive means for research and diagnostics due to its high specificity, precision, sensitivity, versatility, and the ability to develop multiplexed assays for the "absolute" quantitation of virtually any protein target. However, due to the large dynamic range of protein concentrations in blood, high abundance proteins in blood plasma hinder the detectability and quantification of lower-abundance proteins which are often relevant in the context of different diseases. Here we outline a streamlined method involving offline high-pH reversed-phase fractionation of human plasma samples followed by the quantitative analysis of specific fractions using nanoLC-parallel reaction monitoring (PRM) on a Q Exactive Plus mass spectrometer for peptide detection and quantitation with increased sensitivity. Because we use a set of synthetic peptide standards, we can more efficiently determine the precise retention times of the target peptides in the first-dimensional separation and specifically collect eluting fractions of interest for the subsequent targeted MS quantitation, making the analysis faster and easier. An eight-point standard curve was generated by serial dilution of a mixture of previously validated unlabeled ("light") synthetic peptides of interest at known concentrations. The corresponding heavy stable-isotope-labeled standard (SIS) analogues were used as normalizers to account for losses during sample processing and analysis. Using this method, we were able to improve the sensitivity of plasma protein quantitation by up to 50-fold compared to using nanoLC-PRM alone.

A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control.

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12-50 ng mL-1 for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1-14 ng mL-1 for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5-100 000 ng mL-1 for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid-liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.

Absorption, distribution, metabolism, and excretion of [14C]Mefuparib (CVL218), a novel PARP1/2 inhibitor, in rats.

Mefuparib (CVL218) is a novel second-generation poly-ADP-ribose polymerase (PARP) inhibitor for cancer treatment. CVL218 can easily enter the brain. However, the transport mechanism by which CVL218 crosses the blood-brain barrier (BBB) is unknown. (1) [14C] CVL218 metabolism in rats was traced by a liquid scintillation counter and oxidative combustion. (2) Metabolic profiles and metabolites were identified by UHPLC-β-RAM/UHPLC-Fraction Collector and UHPLC-Q Exactive Plus MS. (3) The partition coefficient Kp,uu,brain value was simulated by two strategies. One strategy was using ACD and GastroPlus Software based on the results of intravenous administration pharmacokinetics and plasma protein-binding studies. The reliability was confirmed by comparison with another strategy (brain/plasma distribution study). (1) Rapid drug elimination was observed 24h after intragastric administration. The total cumulative excretion in urine and feces within 168h accounted for 97.15% of the dose. The cumulative radioactive dose recovery in bile was 41.87% within 72h. The drug-related substances were extensively distributed to the tissues within 48h. (2) M8 was the major metabolite in plasma, urine, feces and bile. (3) CVL218 exhibited high brain protein-binding rate (88.16%). The Kp,uu,brain value (8.42) simulated by the simple software strategy was similar to that of the brain/plasma distribution study (7.01). CVL218 is a fast-metabolizing drug and is mainly excreted in feces. The B/P ratio prediction and observation data for CVL218 were consistent. Furthermore, the Kp,uu,brain value indicated that penetration through the BBB might be mediated by uptake transporters.

Identification of Protosappanoside D from Caesalpinia decapetala and Evaluation of Its Pharmacokinetic, Metabolism and Pharmacological Activity.

Protosappanoside D (PTD) is a new component isolated from the extract of Caesalpinia decapetala for the first time. Its structure was identified as protosappanin B-3-O-β-D-glucoside by 1H-NMR, 13C-NMR, 2D-NMR and MS techniques. To date, the pharmacological activities, metabolism or pharmacokinetics of PTD has not been reported. Therefore, this research to study the anti-inflammatory activity of PTD was investigated via the LPS-induced RAW264.7 cells model. At the same time, we also used the UHPLC/Q Exactive Plus MS and UPLC-MS/MS methods to study the metabolites and pharmacokinetics of PTD, to calculate its bioavailability for the first time. The results showed that PTD could downregulate secretion of the pro-inflammatory cytokines. In the metabolic study, four metabolites were identified, and the primary degradative pathways in vivo involved the desaturation, oxidation, methylation, alkylation, dehydration, degradation and desugarization. In the pharmacokinetic study, PTD and its main metabolite protosappanin B (PTB) were measured after oral and intravenous administration. After oral administration of PTD, its Tmax was 0.49 h, t1/2z and MRT(0–t) were 3.47 ± 0.78 h and 3.06 ± 0.63 h, respectively. It shows that PTD was quickly absorbed into plasma and it may be eliminated quickly in the body, and its bioavailability is about 0.65%.

Urinary Aromatic Amino Acid Metabolites Associated With Postoperative Emergence Agitation in Paediatric Patients After General Anaesthesia: Urine Metabolomics Study.

Background: Emergence agitation (EA) is very common in paediatric patients during recovery from general anaesthesia, but underlying mechanisms remain unknown. This prospective study was designed to profile preoperative urine metabolites and identify potential biomarkers that can predict the occurrence of EA. Methods: A total of 224 patients were screened for recruitment; of those, preoperative morning urine samples from 33 paediatric patients with EA and 33 non-EA gender- and age-matched patients after being given sevoflurane general anaesthesia were analysed by ultra-high-performance liquid chromatography (UHPLC) coupled with a Q Exactive Plus mass spectrometer. Univariate analysis and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were used to analyse these metabolites. The least absolute shrinkage and selection operator (LASSO) regression was used to identify predictive variables. The predictive model was evaluated through the receiver operating characteristic (ROC) analysis and then further assessed with 10-fold cross-validation. Results: Seventy-seven patients completed the study, of which 33 (42.9%) patients developed EA. EA and non-EA patients had many differences in preoperative urine metabolic profiling. Sixteen metabolites including nine aromatic amino acid metabolites, acylcarnitines, pyridoxamine, porphobilinogen, 7-methylxanthine, and 5′-methylthioadenosine were found associated with an increased risk of EA, and they all exhibited higher levels in the EA group than in the non-EA group. The main metabolic pathways involved in these metabolic changes included phenylalanine, tyrosine and tryptophan metabolisms. Among these potential biomarkers, L-tyrosine had the best predictive value with an odds ratio (OR) (95% CI) of 5.27 (2.20–12.63) and the AUC value of 0.81 (0.70–0.91) and was robust with internal 10-fold cross-validation. Conclusion: Urinary aromatic amino acid metabolites are closely associated with EA in paediatric patients, and further validation with larger cohorts and mechanistic studies is needed. Clinical Trial Registration: clinicaltrials.gov, identifier NCT04807998

Identification and dipeptidyl peptidase IV (DPP-IV) inhibitory activity verification of peptides from mouse lymphocytes

The objective of this study was to isolate and identify the intracellular bioactive peptides from mouse lymphocytes before and after lipopolysaccharide (LPS) stimulation, to explore novel peptides and to research the bioactive function. Mouse spleen lymphocytes were isolated and cultured with LPS stimulation (experimental group) or not (control group) to collect intracellular peptides. Totally 385 peptides were analyzed by nanoliter liquid phase-Q Exactive quadrupole ultra-high resolution orbitrap mass spectrometer (Nano LC-Q Exactive Plus) and identified by PEAKS X software. After compared with peptides reported, 131 novel peptides were discovered, which then were predicted bioactivity by Peptide Ranker and 6 peptides with high bioactivity were predicted function by BIOPEP-UMW database. Prediction data showed that they may have dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Finally, two peptides showed better potent inhibition were verified with competitive and noncompetitive modes.

Mass Defect Filtering-Oriented Identification of Resin Glycosides from Root of Convolvulus scammonia Based on Quadrupole-Orbitrap Mass Spectrometer.

This work aimed to develop and evaluate a post-acquisition data processing strategy, referred to as a mass defect filter (MDF), for rapid target the resin glycosides in root of Convolvulus scammonia by setting mass rang and mass defect range from high-resolution MS data. The full-scan mass data were acquired by high-performance liquid chromatography coupled with Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer that featured high resolution, mass accuracy, and sensitivity. To screen resin glycosides, three parent filter m/z 871, m/z 853, and m/z 869 combined with diagnostic fragment ions (DFIs) approach were applied to remove the interference from complex herbal extract. The targeted components were characterized based on detailed fragment ions. Using this approach, 80 targeted components, including 22 glycosidic acids and 58 resin glycosides were tentatively identified. The present results suggested that the proposed MDF strategy would be adaptable to the analysis of complex system in relevant filed.

Production and fermentation characteristics of antifungal peptides by synergistic interactions with Lactobacillus paracasei and Propionibacterium freudenii in supplemented whey protein formulations

Whey protein, as a nutrient-rich waste from dairy industry, several studies have investigated its bioconversion. This study aimed to convert whey protein into antifungal peptides by synergistic interactions with Lactobacillus paracasei and Propionibacterium freudenreichii. The response surface methodology was employed for optimization. The optimum fermentation medium contained 26.69 g/L lactose, 28.44 g/L nitrogen sources (soytone: whey protein = 2:1, m/m) and the inoculum size was at 3.72% (v/v). The fermentate was sensitive to proteolytic enzymes, strong alkaline (pH > 10.0) and high temperature. A total of 156 peptides were identified by nanoliter liquid phase-Q Exactive quadrupole ultra-high resolution orbitrap mass spectrometer (Nano LC-Q Exactive Plus) and screened by PEAKS X software. Finally, the decisive factors of antifungal function were determined by principal component analysis. The research provided experimental and theoretical support for the process optimization and mechanistic explanation of whey protein fermentation to produce antifungal substances.

FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.