Chromatography-electrospray Ionization-tandem Mass Spectrometry Research Articles

Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable d-isomers can be introduced as impurities in amino acid starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of a peptide. The peptide is hydrolyzed in deuterated acid to facilitate correction for any racemization occurring during this step of sample preparation, and the amino acids are subsequently separated by chiral chromatography interfaced with ESI-MS/MS for quantitation. The amino acid samples are analyzed directly following hydrolysis using high-low chromatography and extraction of selected ion response, providing efficiency and simplicity by avoiding the derivatization steps and multiple external standards required by traditional methodologies. GMP method validation feasibility is described for all nineteen chiral proteogenic amino acids. The practical application of the chiral HPLC-ESI-MS/MS method was demonstrated through the recovery of d-amino acid substitutions at each residue of an octapeptide across the 0.1–1.0 % range of interest. The method was applied to the analysis of four model peptides, each consisting of 8–14 amino acid residues, and the results were comparable to those provided by traditional testing methods.

Comparative analysis of the metabolites in Pinellia ternata from two producing regions using ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry

Abstract The rhizomes of Pinellia ternata have a long history of being used as a traditional Chinese herb. To determine the chemical constituents of Pinelliae rhizome, Xi banxia and Jing banxia were collected from two different producing regions and subjected to analysis using ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry. A total of 573 metabolites were identified in 12 different categories. Next, cluster analysis and principal component analysis were performed, which revealed a distinct separation between the two species. The analysis of the differential metabolites revealed that among the 155 metabolites in Xi banxia, 68 metabolites were upregulated, and 87 metabolites were downregulated. The relative concentration of flavonoids in Xi banxia was higher than that in Jing banxia, while the relative concentrations of alkaloids, phenolic acids, and terpenoids were higher in Jing banxia. These results would provide a theoretical basis for the pharmacological activity analysis and functional study of P. ternata.

Metabolomics analysis reveals the accumulation patterns of flavonoids and phenolic acids in quinoa (Chenopodium quinoa Willd.) grains of different colors

Quinoa grains are gaining increasing popularity owing to their high nutritional merits. However, only limited information is available on the metabolic profiles of quinoa grains. In this study, we determined the metabolic profiles of black, red, and white quinoa grains via an ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS)-based metabolomics. A total of 689 metabolites were identified, among which 251, 182, and 317 metabolites displayed different accumulation patterns in the three comparison groups (Black vs Red, Black vs White, and Red vs White), respectively. In particular, flavonoid and phenolic acid contents displayed considerable differences, with 22 flavonoids, 5 phenolic acids, and 1 betacyanin being differentially accumulated among the three quinoa cultivars. Additionally, correlation analysis showed that flavonoids and phenolic acids could act as betanin co-pigments in quinoa grains. In conclusion, this study provides comprehensive insights into the adequate utilization and development of novel quinoa-based functional foods.

Fabrication of novel BiPO4/Ag3PO4@rGO hybrid composite for effective detoxification of tetracycline

A practical photocatalytic method using efficient and nontoxic is crucial for wastewater treatment technology. The present study deals with the preparation of BiPO4/Ag3PO4@rGO heterojunction through hydrothermal process and utilized it for efficient degradation and detoxification of Tetracycline (TCL) antibiotic. The prepared composite was characterized by X-ray diffraction, UV–vis DRS spectroscopy, Scanning electron microscope (SEM), Transmission electron microscope (TEM) and XPS (X-ray photoelectron spectroscopy). From our study, it was evident that the addition of Ag3PO4 extensively improved the photocatalytic efficiency of BiPO4 with a degradation of the rate of 94.6% (k = 0.01783 min−1) towards TCL under visible light within 90 min irradiation. The heterojunction energy-band theory has been adopted to understand the mechanism of degradation. The improved efficiency was ascribed to the excellent charge transfer between the interface of p-n heterojunction and the improvement in the absorption of light. Furthermore, LC/ESI-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) carried out TCL degradation product identification to propose the degradation pathway. The biotoxicity assessment studies revealed that effective detoxification was observed during degradation. Thus, this work extends new methods for developing new BiPO4-based heterojunction composites to meet the requirements for remediation of a contaminated aqueous environment.

Development of methods for quantitative determination of the total and reactive polysulfides: Reactive polysulfide profiling in vegetables

Alliaceous and cruciferous vegetables are rich in bioactive organosulfur compounds, including polysulfides, which exhibit a broad spectrum of potential health benefits. Here, we developed novel, accurate, and reproducible methods to quantify the total polysulfide content (TPsC) and the reactive polysulfide content (RPsC) using liquid chromatography–electrospray ionization–tandem mass spectrometry, and analyzed the reactive polysulfide profiles of 22 types of fresh vegetables, including onions, garlic, and broccoli. Quantitative analyses revealed that onions contained the largest amounts of polysulfides, followed by broccoli, Chinese chive, and garlic. A strong positive correlation was observed between the TPsC and RPsC, whereas only a moderate positive correlation was found between the total sulfur content and TPsC. These results suggest that reactive polysulfide profiling can be a novel criterion for evaluating the beneficial functions of vegetables and their derivatives, which may lead to an understanding of the detailed mechanisms underlying their bioactivities.

Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata.

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I-III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

LC–MS/MS Coupled with Chemometric Analysis as an Approach for the Differentiation of Fritillariae cirrhosae Bulbus and Fritillariae pallidiflorae Bulbus

Fritillariae cirrhosae bulbus (FCB) is one of the most important traditional Chinese medicines (TCM) for the treatment of cough and phlegm. Due to increasing demand and the complexity of FCB’s botanical origin, various substitutes have appeared in the market, resulting in a major challenge to distinguish FCB and its substitutes (F. pallidiflorae bulbus, FPB). Therefore, discriminating FCB from FPB has becoming an urgent necessity. In this study, an ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous quantification of nine steroidal alkaloids (imperialine-3-β-D-glucoside, imperialine, verticine, verticinone, peimisine, yibeinoside A, delavine, delavinone, ebeidinone) within 8 min. According to the composition and content of the above nine compounds, multivariate chemometric analyses were applied for the classification of FCB and FPB. The quantitative results showed that there were both similarities and differences in the content of nine steroidal alkaloids between FCB and FPB, and it was difficult to directly distinguish these two species. Fortunately, with the aid of chemometric analyses, FCB and FPB were successfully differentiated by partial least squares discrimination analysis (PLS-DA) and orthogonal partial least squares discrimination analysis (OPLS-DA) models based on the nine alkaloids’ content. Moreover, four compounds (yibeinoside A, ebeiedinone, delavinone and imperialine) were discovered as potential markers for the identification and differentiation of FCB and FPB. Additionally, compared to other studies, this work collected a large number of samples (49 batches of FCB and 17 batches of FPB) to ensure the reliability of the results. In conclusion, this work established a new approach for the authentication of FCB based on its active components, which provides a good reference for the quality control of FCB and will help us to understand the chemical composition differences between FCB and its adulterants further.

Ecological Risks of Antibiotics in Urban Wetlands on the Qinghai-Tibet Plateau, China.

Although the ecological risks of antibiotics have been extensively researched globally, fewer studies have been conducted in sensitive and fragile plateau wetland ecosystems. To evaluate the ecological risk of antibiotics in plateau urban wetlands, 18 water samples, 10 plant samples, and 8 sediment samples were collected in March 2022 in the Xining urban wetlands on the Qinghai-Tibet Plateau. The liquid chromatography-electrospray ionization tandem mass spectrometry method was utilized to measure the concentrations of 15 antibiotics in three categories in three types of environmental media. Risk quotients were adopted to assess the ecological risk of antibiotics, and the principal component analysis-multiple linear regression model was used to analyze the source of antibiotics. The results showed that (1) the maximum concentrations of antibiotics in water samples, plants, and sediments reached 1220.86 ng/L, 78.30 ng/g, and 5.64 ng/g, respectively; (2) Tylosin (TYL), norfloxacin (NFX), ofloxacin (OFX), and ciprofloxacin (CFX) in water were at medium and high-risk levels, and OFX had the highest risk value, of 108.04; and (3) the results of source apportionment indicate that 58.94% of the antibiotics came from the Huangshui river and wastewater treatment plant (WWTP) near the wetlands. The current study may provide a reference for the risks and management of antibiotics in plateau urban wetlands.

Combined widely targeted metabolomics and transcriptomics analysis reveals differentially accumulated metabolites and the underlying molecular bases in fleshy taproots of distinct radish genotypes

Radish is an important taproot crop with medicinal and edible uses that is cultivated worldwide. However, the differences in metabolites and the underlying molecular bases among different radish types remain largely unknown. In the present study, a combined analysis of liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and RNA-Seq data was conducted to uncover important differentially accumulated metabolites (DAMs) among radish accessions with green, white and red taproot flesh colours. A total of 657 metabolites were identified and 138 DAMs were commonly present in red vs. green and red vs. white accessions. Red accessions were rich in phenolic compounds, while green and white accessions had more amino acids. Additionally, 41 metabolites and 98 genes encoding 37 enzymes were enriched in the shikimate and phenolic biosynthesis pathways. CHS is the rate-limiting enzyme determining flavonoid differences among accessions. A total of 119 candidate genes might contribute to red accession-specific accumulated metabolites. Specifically, one gene cluster consisting of 16 genes, including one RsMYB1, RsMYBL2, RsTT8, RsDFR, RsANS, Rs4CL3, RsSCPL10, Rs3AT1 and RsSAP2 gene, two RsTT19 and RsWRKY44 genes and three RsUGT genes, might be involved in anthocyanin accumulation in red radish fleshy taproots. More importantly, an InDel marker was developed based on an RsMYB1 promoter mutation, and the accuracy reached 95.9% when it was used to select red-fleshed radishes. This study provides comprehensive insights into the metabolite differences and underlying molecular mechanisms in fleshy taproots among different radish genotypes and will be beneficial for the genetic improvement of radish nutritional quality.

Influence of mobile phase composition on the analytical sensitivity of LC–ESI–MS/MS for the concurrent analysis of bisphenols, parabens, chlorophenols, benzophenones, and alkylphenols

Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a “dilute and shoot” approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.

Application of a three dimensional polyethyleneimine functionalized graphene oxide aerogel as an adsorbent for the determination of phytohormones in ginseng.

Aerogels have attracted considerable attention in sample pretreatment for their outstanding properties, such as the unique porous structure, large surface area and abundant modifiable active sites. The present research reports a three-dimensional interconnected porous network aerogel (PEI-AGO) manufactured based on graphene oxide (GO), polyethyleneimine (PEI) and agar as basic materials through a vacuum freeze-drying treatment. The PEI-AGO aerogel exhibits great potential as a solid phase extraction adsorbent for the selective purification of six endogenous plant hormones in conjunction with high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS). Several factors affecting the extraction efficiency were investigated. Under the optimized extraction conditions, a wide linear range of 0.5-100 ng mL-1 with a good linearity (r > 0.9934) was observed. Low limits of detection (LODs) and limits of quantification (LOQs) were obtained in the range of 0.032-0.155 ng mL-1 and 0.107-0.518 ng mL-1, respectively. Furthermore, the relative recoveries for spiked ginseng samples exhibited remarkable consistency, ranging from 90.2% to 117.6%, with a relative standard deviation (RSD) of ≤9.4% (n = 3). In summary, PEI-AGO has proven to be an effective adsorbent for the pretreatment and enrichment of phytohormones which can be used for the determination of trace endogenous acidic plant hormones in ginseng leaves.

Transcription and Metabolism Pathways of Anthocyanin in Purple Shamrock (Oxalis triangularis A.St.-Hil.).

Anthocyanins are water-soluble pigments that can impart various colors to plants. Purple shamrock (Oxalis triangularis) possesses unique ornamental value due to its purple leaves. In this study, three anthocyanins, including malvidin 3-O-(4-O-(6-O-malonyl-glucopyranoside)-rhamnopyranosyl)-5-O-(6-O-malonyl-glucopyranoside), delphinidin-3-O-rutinoside and malvidin-3,5-di-O-glucoside, were characterized with ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in purple shamrock. To investigate the molecular mechanism of anthocyanin biosynthesis in green shamrock (Oxalis corymbosa) and purple shamrock, RNA-seq and qRT-PCR were performed, and the results showed that most of the anthocyanin biosynthetic and regulatory genes were up-regulated in purple shamrock. Then, dark treatment and low temperature treatment experiments in purple shamrock showed that both light and low temperature can induce the biosynthesis of anthocyanins.

Biodegradation of Punicalagin into Ellagic Acid by Selected Probiotic Bacteria: A Study of the Underlying Mechanisms by MS-Based Proteomics.

Pomegranate (Punica granatum L.) is a well-known source of bioactive phenolic compounds such as ellagitannins, anthocyanins, and flavanols. Punicalagin, one of the main constituents of pomegranate, needs to be biodegraded by bacteria to yield metabolites of medicinal interest. In this work, we tested 30 lactic acid bacteria (LAB) and their capacity to transform punicalagin from a punicalagin-rich pomegranate extract into smaller bioactive molecules, namely, ellagic acid and urolithins. These were identified and quantified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS2). Further, we evaluated the molecular mechanism governing this transformation through label-free comparative MS-based proteomics. All tested LAB strains were capable of transforming punicalagin into ellagic acid, while the biosynthesis of urolithins was not observed. Proteomic analysis revealed an increase of generic transglycosylases that might have a hydrolytic role in the target phenolic molecule, coupled with an increase in the quantity of ATP-binding cassette (ABC) transporters, which might play a relevant role in transporting the resulting byproducts in and out of the cell.

Evaluation of a Capillary Microsampling Device for Analyzing Plasma Lenvatinib Concentration in Patients With Hepatocellular Carcinoma.

The anticancer drug, Lenvima (lenvatinib), has severe side effects. Therapeutic drug monitoring helps ensure its efficacy and safety. Regular and optimally timed blood sampling is tough, especially when lenvatinib is self-medicated. Microsampling using the easy to handle Microsampling Wing (MSW) may help circumvent this problem. However, current lenvatinib detection methods are not sensitive enough to detect its concentrations in microsamples (<50-250 μL). Thus, the aim of this study was 2-fold (1) develop an analytic method to estimate plasma lenvatinib concentrations in microsamples and (2) verify whether this method works on micro (5.6 μL) blood plasma samples obtained clinically through MSW from patients with unresectable hepatocellular carcinoma (HCC). A simple, highly sensitive, and specific liquid chromatography-electrospray ionization tandem mass spectrometry method was developed. Using this novel protocol, the trough blood plasma concentration of lenvatinib was measured for both blood sampled conventionally and that using MSW. Thirty-five venous whole blood samples were obtained from 11 patients with HCC. Furthermore, the stability of lenvatinib in MSW samples during storage was evaluated. The mean plasma lenvatinib concentration estimates were not significantly different between the MSW and conventional venous blood samples. CV for interday and intraday assays was low. Up to day 5, the lenvatinib concentration in the MSW samples was 85%-115% of the initial day concentration (when stored at 25°C or 4°C). The interference of endogenous matrix components in the human plasma was low. These results indicate that the novel mass spectrometry protocol accurately measures lenvatinib in human plasma and is reproducible. Thus, MSW could be a useful microsampling device for lenvatinib therapeutic drug monitoring in patients with HCC when used in combination with this novel liquid chromatography-electrospray ionization tandem mass spectrometry detection method.

Lipidomic analysis and triglyceride profiles of fish oil: Preparation through silica gel column and enzymatic treatment

A comprehensive study of lipidomic coupled with triglyceride profiles onto four fish oils was performed through ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Overall, 1010 lipids belonging to 6 categories and 38 lipid classes were identified. Triglycerides (TGs) were the dominant component in four fish oils (40 %–99 % of total lipids), and glycerophospholipids (GPs) and sphingolipids (SLs) were another two major lipid categories in the fish oil (TG50) which prepared through silica gel column. These results revealed that enzymatic treatment has slight effect on lipid distribution but silica gel column could change the lipids composition. TGs composition of four fish oils were separated completely, and the most TG molecule in TG50 is TG(18:3_14:0_18:0), possessed 13.03 ± 5.07 % relative content, these results implied that silica gel column could protect the nature structure of TGs from destroying which may also limited to further improve eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) purity, but enzymic method was not restricted by this.

Studies on latent and soluble polyphenol oxidase from Moringa oleifera Lam. leaves

Polyphenol oxidase (PPO) from Moringa oleifera leaves was investigated to unravel its unique properties that can be exploited for possible biotechnological application(s).Soluble and latent PPO were extracted and purified to homogeneity using a combination of three-phase partitioning and gel filtration chromatography. The purity and molecular weight of the purified protein were estimated by SDS-PAGE and further by nano liquid chromatography-electrospray ionization tandem mass spectrometry (Nano-LC-ESI-MS/MS) and amino acid composition determination. Physicochemical and kinetic properties of the purified Moringa oleifera PPO (moPPO) were investigated following established protocols.Two soluble isoforms and a latent form of PPO were purified from the leaves of Moringa oleifera. All purified proteins were monomeric with molecular weights ranging from 10 to 65 kDa. PPO with the lowest molecular weight was further confirmed to be 7.6 kDa, 10.5 ± 0.06 kDa 7.2 kDa, and 8.61 ± 1.69 kDa as estimated by Nano-LC-ESI-MS/MS, SDS-PAGE, amino acid composition determination, and SEC respectively. The soluble proteins have pH optima at pH 6.5 and slightly basic pH 8.0, and that of the latent PPO was found to be 5.5 and 8.0 respectively. The optimal temperature for the purified enzymes was between 15 ᵒC – 25ᵒC and their catalytic efficiency was between 0.4 - 1.9 x 105 M−1s−1.The study concluded that M. oleifera leaves contain at least three forms of PPO, a novel low molecular weight peptide enzyme, a soluble form and a latent form with high kcat/Km similar to those obtainable from other sources which could be of interest in biotechnological applications.

Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus Strains from Dairy and Fermented Food Products.

Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors attach to host cells and are also involved in immune evasion. LC-MS/MS-based methods offer several advantages compared with other approaches because one can directly identify microbial peptides without the necessity of inferring conclusions based on other approaches such as genomics tools. The present study describes the use of liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) to perform a global shotgun proteomics characterization for opportunistic pathogenic Enterococcus from different dairy and fermented food products. This method allowed the identification of a total of 1403 nonredundant peptides, representing 1327 proteins. Furthermore, 310 of those peptides corresponded to proteins playing a direct role as virulence factors for Enterococcus pathogenicity. Virulence factors, antibiotic sensitivity, and proper identification of the enterococcal strain are required to propose an effective therapy. Data are available via ProteomeXchange with identifier PXD036435. Label-free quantification (LFQ) demonstrated that the majority of the high-abundance proteins corresponded to E. faecalis species. Therefore, the global proteomic repository obtained here can be the basis for further research into pathogenic Enterococcus species, thus facilitating the development of novel therapeutics.

Chemical Characterization of a Collagen-Derived Protein Hydrolysate and Biostimulant Activity Assessment of Its Peptidic Components.

Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography–electrospray ionization–tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH.

Determination of the Oxidative Stress Biomarkers of 8-Hydroxydeoxyguanosine and Dityrosine in the Gills, Skin, Dorsal Fin, and Liver Tissue of Atlantic Salmon (Salmo salar) Parr.

Oxidative stress is a condition caused by an imbalance in the occurrence of reactive oxygen species in the cells and tissues of organisms. An ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous determination of two oxidative stress biomarkers, 8-hydroxydeoxyguanosine (8OHDG) and dityrosine (DIY), in the gills, skin, dorsal fin, and liver tissue of Atlantic salmon (Salmo salar) parr. The use of target analyte-specific 13C and 15N internal standards allowed quantification of each target analyte to be performed through the standard solvent calibration curve. The relative recoveries [mean ± (relative standard deviation%)] of 8OHDG and DIY were 101 ± 11 and 104 ± 13% at a fortified concentration of 10 ng/mL (8OHDG) and 500 ng/mL (DIY), respectively, ensuring the accuracy of the extraction and quantification. The chromatographic separation was carried out using a gradient elution program with a total run time of 5 min. The limits of detection (LODs) were 0.11 and 1.37 ng/g wet weight (w.w.) for 8OHDG and DIY, respectively. To demonstrate the applicability of the developed method, it was applied in 907 tissue samples that were collected from Atlantic salmon parr individuals reared in an experimental land-based recirculating aquaculture system (RAS) treated with peracetic acid. Moreover, the possibility of using the dorsal fin as an alternative matrix for the minimally invasive assessment of oxidative stress in Atlantic salmon parr was introduced. To our knowledge, 8OHDG and DIY were used for the first time as biomarkers for biomonitoring the fish health (oxidative stress) of Atlantic salmon parr in RAS.

Divergent substrate specificities and regioselectivities of three lipase isoforms from Cordyceps militaris: Combinatorial advantages for entomopathogenicity and prospects as biocatalysts

Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60–70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography–electrospray ionization–tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.