Chromatography-electrospray Ionization-tandem Mass Spectrometry Research Articles

Plasma ceramides are associated with outcomes in acute ischemic stroke patients

Sphingolipids are major constituents of eukaryotic cell membranes and play key roles in cellular regulatory processes. Our recent results in an experimental stroke animal model demonstrated changes in sphingolipids in response to acute ischemic brain injury. This study aimed to investigate the plasma levels of sphingosine-1-phosphate (S1P) and ceramides in acute ischemic stroke (AIS) patients and their associations with functional outcomes. Plasma samples were collected from patients with AIS at <48 and 48-72h post stroke and from nonstroke controls. The levels of S1P and ceramides with different fatty acyl chain lengths were measured by the ultra-high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). A poor functional outcome was defined as a modified Rankin Scale (mRS) score ≥2 at 3 months after AIS. The results showed that S1P and very-long-chain ceramides were significantly decreased in AIS patients (n=87; poor outcome, 56.3%) compared to nonstroke controls (n=30). In contrast, long-chain ceramides were significantly increased in AIS patients. More importantly, higher levels of Cer(d18:1/18:0), Cer(d18:1/20:0), and Cer(d18:1/22:0) at 48-72h were significantly associated with poor functional outcomes after adjusting for potential clinical confounders, including age, sex, hypertension, and National Institutes of Health Stroke Scale score at admission. Our study supported the dynamic metabolism of sphingolipids after the occurrence of AIS. Ceramides could be potential prognostic markers for patients with AIS.

A novel isotope dilution UHPLC-ESI-MS/MS method for the quantification of 3-monochloropropane-1,2-diol in Caco-2 cell transport and receiving buffers

ABSTRACT A routine, selective and sensitive ultra-high performance liquid chromatography-electrospray ionisation tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the quantification of 3-monochloropropane-1,2-diol (3-MCPD) in Caco-2 cell transport buffer (FaSSIF-V2, the second version of a fasted state simulated intestinal fluid) and receiving buffer (HBSS, Hank’s balanced salt solution). The method involves measuring deuterated 3-MCPD (3-MCPD-d5) as internal standard (IS) during the entire analytical procedure to obtain precise and accurate results. The separation was performed on a Poroshell 120 HILIC column (2.7 µm, 3.0 × 50 mm) at a flow rate of 0.3 mL/min using water (containing 0.025% acetic acid) and acetonitrile (containing 0.025% acetic acid) as the mobile phases. Mass spectrometric detection was operated in dynamic multiple reaction monitoring (dMRM) in negative ion mode. The method exhibited high sensitivity. The limits of detection (LOD) for 3-MCPD in FaSSIF-V2 and HBSS were 0.012 and 0.014 µmol/L, and the limits of quantification (LOQ) were 0.039 and 0.045 µmol/L, respectively. Satisfactory results were observed for linearity (R2 > 0.999), intra-day precision (RSD% <7.7% in FaSSIF-V2 and <6.6% in HBSS), inter-day precision (RSD% <5.9% in FaSSIF-V2 and <5.6% in HBSS), accuracy (% error within ± 10%), and sample stability (RSD% <7.7% and % error within ± 10%). The method has been successfully applied to quantify 3-MCPD in Caco-2 cell transport and receiving buffers. The results were in good agreement with those obtained with gas chromatography-tandem mass spectrometry (GC-MS).

Simultaneous quantification of cefuroxime and clindamycin in human lumbar anulus fibrosus, nucleus pulposus and serum via UPLC-MS/MS

BackgroundThis study aimed to develop a sensitive, accurate method for simultaneously quantifying cefuroxime and clindamycin in human serum, lumbar anulus fibrosus and nucleus pulposus. MethodsCefuroxime and clindamycin were quantified using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode on a triple-quadrupole AB Qtrap 5500 system in positive ion mode. Internal standards were D3-cefuroxime and D3,13C-clindamycin. Samples were pretreated by precipitating total protein. ResultsThe method showed high sensitivity and good linearity over broad calibration ranges from 100 to 100 000 ng/mL for cefuroxime and 10 to 10 000 ng/mL for clindamycin in serum, and from 10 to 10 000 ng/mL for cefuroxime and 1 to 1 000 ng/mL for clindamycin in lumbar nucleus pulposus. In all sample types, correlation coefficients were greater than 0.99, intra- and inter-day precision (relative standard deviation) was less than 15%, and accuracy (relative error) was within 14% for both analytes. This method was effective at quantifying penetration of cefuroxime and clindamycin in patients undergoing oblique lumbar interbody fusion surgery. ConclusionsA very sensitive, specific method for simultaneous detection of cefuroxime and clindamycin has been developed for human lumbar anulus fibrosus, nucleus pulposus and serum samples.

Identification and characterization of in vitro and in vivo fidarestat metabolites: Toxicity and efficacy evaluation of metabolites.

The progression of diabetic complications can be prevented by inhibition of aldose reductase and fidarestat considered to be highly potent. To date, metabolites of the fidarestat, toxicity, and efficacy are unknown. Therefore, the present study on characterization of hitherto unknown in vitro and in vivo metabolites of fidarestat using liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) is undertaken. In vitro and in vivo metabolites of fidarestat have been identified and characterized by using LC/ESI/MS/MS and accurate mass measurements. To identify in vivo metabolites, plasma, urine, and feces samples were collected after oral administration of fidarestat to Sprague-Dawley rats, whereas for in vitro metabolites, fidarestat was incubated in human S9 fraction, human liver microsomes, and rat liver microsomes. Furthermore, in silico toxicity and efficacy of the identified metabolites were evaluated. Eighteen metabolites have been identified. The main in vitro phase I metabolites of fidarestat are oxidative deamination, oxidative deamination and hydroxylation, reductive defluroniation, and trihydroxylation. Phase II metabolites are methylation, acetylation, glycosylation, cysteamination, and glucuronidation. Docking studies suggest that oxidative deaminated metabolite has better docking energy and conformation that keeps consensus with fidarestat whereas the rest of the metabolites do not give satisfactory results. Aldose reductase activity has been determined for oxidative deaminated metabolite (F-1), and it shows an IC50 value of 0.44 μM. The major metabolite, oxidative deaminated, did not show any cytotoxicity in H9C2, HEK, HEPG2, and Panc1 cell lines. However, in silico toxicity, the predication result showed toxicity in skin irritation and ocular irritancy SEV/MOD versus MLD/NON (v5.1) model for fidarestat and its all metabolites. In drug discovery and development research, it is distinctly the case that the potential for pharmacologically active metabolites must be considered. Thus, the active metabolites of fidarestat may have an advantage as drug candidates as many drugs were initially observed as metabolites.

Shotgun Proteomics for Food Microorganism Detection.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

1H NMR-based metabolomics and UHPLC-ESI-MS/MS for the investigation of bioactive compounds from Lupinus albus fractions

Lupinus albus or white lupine has recently received increase attention for its medicinal values. Several studies have described the hypoglycemic effect of the white lupine, which is known as a food plant with potential value for treatment of diabetes. This study provides useful information for the identification and quantification of compounds in L. albus fractions by proton nuclear magnetic resonance (1H NMR) spectroscopy. In total, 35 metabolites were identified from L. albus fractions.Principal component analysis (PCA) was used as a multivariate projection method for visualizing the different composition of four different fractions. The bioactivities of fractions with different polarity obtained from the extract of L. albus seeds are reported. Among the fractions studied, the chloroform fraction (CF) exhibits a high free radical scavenging (DPPH) and α-glucosidase inhibitory activities with IC50 values of 24.08 and 20.08 μg/mL, respectively. A partial least-squares analyses (PLS) model had been successfully performed to correlate the potential active metabolites with the corresponding biological activities. Metabolites containing proline, caprate, asparagine, lupinoisolone C, hydroxyiso lupalbigenin and some unknown compounds show high correlation with the bioactivities studied. Moreover, the structural identification in the active fraction was supported by ultrahigh-performance-liquid chromatography–electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) analysis. A total of 21 metabolites were tentatively identified from MS/MS data by comparison with previously reported data. Most of these compounds are isoflavonoids without known biological activity. This information may be useful for developing functional food from L. albus with potential application in the management of diabetes.

Quantitative MALDI-MS assay of steroid hormones in plasma based on hydroxylamine derivatization

Measuring the concentrations of steroid hormones in plasma is critical for understanding their role in various vital physiological processes. The detection of underivatized steroid hormones in biofluids through mass spectrometry (MS) is typically hindered by low ionization efficiency. We described a novel matrix-assisted laser desorption/ionization-MS (MALDI-MS) approach based on hydroxylamine derivatization (HA-D) to analyze low-concentration steroid hormones in plasma. The ketonic carbonyl group containing steroid hormones could be derivatized using HA to form oxime derivatives, which considerably enhanced the MS sensitivity for detecting steroid hormones. By using the optimized conditions, estrone (E1), testosterone (T), and progesterone (Prog), could be simultaneously quantified in plasma with a limit of detection (LOD) from 0.019 to 0.031 nM, recoveries from 86% to 108%, and coefficient of variation (CV%) from 4.59% to 11.90%. HA-D/MALDI-MS exhibited higher sensitivity than those using Girard T (GT). To establish potential utility of our method, we characterized fatty liver patient plasmas to demonstrate that the HA-D/MALDI-MS procedure could generate quantitative results comparable to the current clinical liquid chromatography-electrospray ionization tandem MS (LC-ESI MS/MS) method. This approach facilitates the rapid and accurate characterization of plasma hormones, and renders the MALDI-MS approach for steroid hormones more adaptable for clinical research and use.

Fully automated sample treatment method for high throughput proteome analysis

The bottom-up strategy for proteome analysis typically employs a multistep sample preparation workflow that suffers from being time-consuming and sample loss or contamination caused by the off-line manual operation. Herein, we developed a hollow fibre membrane (HFM)-aided fully automated sample treatment (FAST) method. Due to the confinement effects of HFMs and the immobilized enzymatic reactor, the proteome samples could be denatured, reduced, desalted and digested within 8–20 min via the one-stop service. This method also showed superiority in trace sample analysis. In one and half hours, we could identify about 1,600 protein groups for 500 HeLa cells as the starting materials, 1.5–8 times more than those obtained by previously reported methods. Through the on-line combination of FAST with nano-liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS), we further established a fully integrated platform for label-free quantification of proteome with high reproducibility and precision. Collectively, FAST presented here represents a major advance in the high throughput sample treatment and quantitative analysis of proteomes.

Preparation of zirconium arsenate-modified monolithic column for selective enrichment of phosphopeptides.

Protein phosphorylation is a crucial posttranslational modification for the regulation of many different biological functions. Selective enrichment of phosphopeptides from the complex biological samples is an essential step for the mass spectrometry analysis of protein phosphorylation. In this study, an arsenate functionalized monolithic column was first prepared by a single-step copolymerization of p-methacryloylaminophenylarsonic acid and ethylene dimethacrylate. Then the metal ions Zr4+ were attached onto the prepared monolithic column via metal-chelate complex formation by Zr4+ and arsenate groups. The obtained monolithic column was employed as a new sorbent for the phosphopeptide enrichment via immobilized metal affinity chromatography. Phosphopeptides analysis was realized by polymer monolith microextraction using this monolithic column coupled to both matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry. The proposed method exhibited a high selectivity for phosphopeptide enrichment in complex matrices, and was applied to the analysis of phosphopeptides in human serum and tryptic digests of rat brain proteins. Four phosphopeptides could be selectively captured from human serum and 2608 endogenous phosphopeptides were identified from the tryptic digests of rat brain proteins, indicating a satisfactory performance of this method for the enrichment of phosphopeptides from complex biological samples.

Metabolism and pharmacokinetics of a novel polyphenol fatty acid ester phloridzin docosahexaenoate in Balb/c female mice

Flavonoids are known to undergo phase II metabolism and produce metabolites with similar or stronger biological effects compared to the parent flavonoids. However, the limited cellular uptake and bioavailability restrict their clinical use. We synthesized phloridzin docosahexaenoate (PZ-DHA), a novel fatty acid ester of polyphenol, through an acylation reaction with the aim of increasing the cellular availability and stability of the parent biomolecules, phloridzin (PZ) and docosahexaenoic acid (DHA). Here, we report metabolites and pharmacokinetic parameters of PZ-DHA, determined using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. PZ-DHA was taken-up by human (MDA-MB-231, MDA-MB-468, and MCF-7) and mouse (4T1) mammary carcinoma and human non-malignant mammary epithelial cells (MCF-10A) in cellular uptake assays. Our results suggested that the acylation improves the cellular uptake of PZ and stability of DHA within cells. In mouse hepatic microsomal assays, two major glucuronides of PZ-DHA, PZ-DHA-4-O-glucuronide and PZ-DHA-4′-O-glucuronide (MW = 923.02 g/mol), were detected. One tri-methylated- (4,4′,6′-O-trimethyl-PZ-DHA) (MW = 788.88 g/mol) and one di-sulphated- (PZ-DHA-4,4′-O-disulphide) PZ-DHA metabolite (MW = 906.20 g/mol) were also identified. Intraperitoneal injections of PZ-DHA (100 mg/kg) into Balb/c female mice was rapidly absorbed with a serum Cmax and Tmax of 23.7 µM and 60 min, respectively, and rapidly eliminated (t1/2 = 28.7 min). PZ-DHA and its metabolites are readily distributed throughout the body (Vd = 57 mL) into many organs. We identified in vitro and in vivo metabolites of PZ-DHA, which could be tested for potential use to treat diseases such as cancer in multiple organ systems.

Determination and pharmacokinetic analysis of ticarcillin disodium-clavulanate potassium for injection in rat plasma by UPLC-ESI-MS/MS.

ObjectiveTo establish a specific and rapid ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS) method for measuring ticarcillin and clavulanate levels in rat plasma.MethodsA Waters ACQUITY BEH C18 column (50 mm × 2.1 mm, 1.7 μm) and SCIEX QTRAP® LC-MS/MS System were used. Analyses were conducted to optimize the chromatographic and MS conditions, and the pharmacokinetic parameters of ticarcillin and clavulanate were assessed.ResultsLinear relationships were observed in the ranges of 10 to 10,000 ng/mL for ticarcillin R (r2 = 0.9967) 30 to 10,000 ng/mL for ticarcillin S (r2 = 0.9961), and 30 to 10,000 ng/mL for clavulanate (r2 = 0.9981). The average extraction recoveries of all compounds ranged from 86.9% to 96.4%. The pharmacokinetic parameters of the ticarcillin R and S isomers in rats were distinctive. The ticarcillin R and S isomers and clavulanate were rapidly absorbed in vivo. Ticarcillin S and clavulanate had similar elimination rates, whereas that of ticarcillin R was slower.ConclusionA UPLC-ESI-MS/MS method was developed and validated for the determination of ticarcillin and clavulanate in rat plasma.

Deciphering the Pharmacological Mechanisms of Ma Xing Shi Gan Decoction against COVID-19 through Integrating Network Pharmacology and Experimental Exploration.

The outbreak of new infectious pneumonia caused by SARS-CoV-2 has posed a significant threat to public health, but specific medicines and vaccines are still being developed. Traditional Chinese medicine (TCM) has thousands of years of experience in facing the epidemic disease, such as influenza and viral pneumonia. In this study, we revealed the efficacy and pharmacological mechanism of Ma Xing Shi Gan (MXSG) Decoction against COVID-19. First, we used liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to analyze the chemical components in MXSG and identified a total of 97 components from MXSG. Then, the intervention pathway of MXSG based on these components was analyzed with network pharmacology, and it was found that the pathways related to the virus infection process were enriched in some of MXSG component targets. Simultaneously, through literature research, it was preliminarily determined that MXSG, which is an essential prescription for treating COVID-19, shared the feature of antiviral, improving clinical symptoms, regulating immune inflammation, and inhibiting lung injury. The regulatory mechanisms associated with its treatment of COVID-19 were proposed. That MXSG might directly inhibit the adsorption and replication of SARS-CoV-2 at the viral entry step. Besides, MXSG might play a critical role in inflammation and immune regulatory, that is, to prevent cytokine storm and relieve lung injury through toll-like receptors signaling pathway. Next, in this study, the regulatory effect of MXSG on inflammatory lung injury was validated through transcriptome results. In summary, MXSG is a relatively active and safe treatment for influenza and viral pneumonia, and its therapeutic effect may be attributed to its antiviral and anti-inflammatory effects.

Mass Spectral Analysis of Synthetic Peptides: Implications in Proteomics.

Sequence determination of peptides is a crucial step in mass spectrometry-based proteomics. Peptide sequences are determined either by database search or by de novo sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics. Developing standards for evaluating the performance of mass spectrometers and algorithms used for identification of proteins is important for proteomics studies. The current study is focused on these aspects by using synthetic peptides. A total of 599 peptides were designed from in silico tryptic digest with 1 or 2 missed cleavages from 199 human proteins, and synthetic peptides corresponding to these sequences were obtained. The peptides were mixed together, and analysis was carried out using liquid chromatography-electrospray ionization tandem mass spectrometry on a Q-Exactive HF mass spectrometer. The peptides and proteins were identified with SEQUEST program. The analysis was carried out using the proteomics workflows. A total of 573 peptides representing 196 proteins could be identified, and a spectral library was created for these peptides. Analysis parameters such as "no enzyme selection" gave the maximum number of detected peptides as compared with trypsin in the selection. False discoveries could be identified. This study highlights the limitations of peptide detection and the need for developing powerful algorithms along with tools to evaluate mass spectrometers and algorithms. It also shows the limitations of peptide detection even with high-end mass spectrometers. The mass spectral data are available in ProteomeXchange with accession no. PXD017992.

Markers of Local and Systemic Estrogen Metabolism in Endometriosis.

Estrogen metabolites (EMs) can work independently from their parent hormones. We hypothesize that in endometriosis, estrogen is metabolized preferentially along hormonally active pathways. We recruited 62 women with endometriosis (proven laparoscopically and histologically) and 52 control women (normal findings with laparoscopy) among patients undergoing surgery for pelvic pain and/or infertility during the proliferative phase of the menstrual cycle. Urinary samples were collected preoperatively. Biopsies from eutopic endometrium of control women and women with endometriosis were collected during surgery. EMs in urine and endometrial tissues were extracted and determined using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). These included: 2-hydroxyestrone (2OHE1), 16-α hydroxyestrone (16α-OHE1), 2OHE1/16α-OHE1 ratio, 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), and 4-hydroxyestradiol (4OHE2). Eutopic endometrium of endometriosis patients, as compared to control endometrium, contained significantly higher level of 4OHE1 (0.03 (IQR: 0.03-0.265) versus 0.03 (IQR: 0.03-0.03) μg/g, respectively, P = 0.005), 2-OHE2 (0.241 (IQR: 0.1-0.960) versus 0.1 (IQR: 0.1-0.1) μg/g, respectively, P < 0.001), and 4-OHE2 (0.225 (IQR: 0.22-1.29) versus 0.0.2 (IQR: 0.2-0.2) μg/g, respectively, P < 0.001). Only 2OHE1 showed higher concentration in urine of women with endometriosis than controls (9.9 (IQR: 3.64-14.88) versus 4.5 (IQR: 1.37-17.00) μg/mg creatinine, respectively, P = 0.042). Eutopic endometrium of women with endometriosis metabolizes estrogen preferentially to the biologically active 2OHE2, and potentially genotoxic 4OHE1 and 4OHE2 metabolites. This contributes to further understanding of endometriosis etiology, its link to ovarian cancer, and could help identifying an endometrial biomarker of the disease.

A Comparative Metabolomics Study of Flavonoids in Radish with Different Skin and Flesh Colors (Raphanus sativus L.).

Radish (Raphanus sativus) is an important worldwide vegetable with a wide variety of colors that affect its appearance and nutritional quality. However, the large-scale detection, identification, and quantification of flavonoids in multicolor radish have rarely been studied. To uncover the diversity and accession-specific flavonoids in radish, liquid chromatography electrospray ionization-tandem mass spectrometry was used to analyze the metabolic profiles in the skin and flesh of six colored radish accessions: light-red Manshenhong, dark-red Touxinhong (TXH), purple Zijinling (ZJL), Xinlimei with red flesh (XLMF) and green skin, white Shizhuangbai (SZB), and black radish. In total, 133 flavonoids, including 16 dihydroflavones, 44 flavones, 14 flavonoids, 9 anthocyanins, and 28 flavonols, were characterized. The flavonoid metabolic profiles differed among the different colored radishes. Red and purple radishes contained similar anthocyanin compounds responsible for color pigmentation, including red cyanidin, callistephin, and pelargonin. Purple ZJL was most enriched with cyanidin o-syringic acid and cyanin, whereas callistephin and pelargonin were more abundant in dark-red TXH. Additionally, the black and white radishes shared similar anthocyanin and flavonoid profiles, suggesting that the color of black radishes was not caused by anthocyanin but by other metabolites. The metabolites in colored radishes that differed from SZB were mainly involved in the biosynthesis of plant secondary metabolites, such as flavonoid, flavone, flavonol, isoflavonoid, and phenylpropanoid biosynthesis. This study provides new insights into the differences in metabolite profiles among radishes with different skin and flesh colors. The results will be useful for aiding the cultivation of valuable new radish varieties.

Rapid determination of thyroid hormones in blood plasma from Glaucous gulls and Baikal seals by HybridSPE®-LC-MS/MS

A rapid hybrid solid phase extraction (HybridSPE®) protocol tailored to liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) analysis, was developed for the determination of four thyroid hormones, L-Thyroxine (T4), 3,3′,5-triiodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3) and 3,3′-diiodo-L-thyronine (T2) in blood plasma from Glaucous gulls (Larus hyperboreus) and Baikal seals (Phoca sibirica). The use of target analyte specific 13C internal standards allowed quantification to be performed through the standard solvent calibration curves and alleviated the need to perform quantification with matrix match curves. The relative recoveries were 100.0–110.1 % for T4, 99.1–102.2 % for T3, 100.5–108.0 % for rT3, and 100.5–104.6 % for T2. The matrix effects ranged from −1.52 to −6.10 %, demonstrating minor signal suppression during analysis. The method intra-day precision (method repeatability, RSD %, N = 5, k = 1 day) and inter-day precision (method reproducibility, RSD %, N = 10, k = 2 days) at the 1 ng/mL concentration of fortification were 8.54–15.4 % and 15.4–24.8 %, respectively, indicating acceptable chromatographic peak stabilities for all target THs even at trace level concentrations. The method limit of detection (LOD) for T4, T3, rT3 and T2 was 0.17, 0.16, 0.30 and 0.17 ng/mL, respectively. The HybridSPE® protocol was simple and rapid (<1 min) upon application, while the HybridSPE® cartridge did not require (as in classical SPE cartridges) any additional equilibration nor conditioning step prior sample loading. A total of 46 blood plasma samples, 30 samples collected from Glaucous gulls and 16 samples collected from Baikal seals, were analyzed for thyroid hormones to demonstrate the applicability of the developed method in these wildlife species. The concentrations of T4 and T3 in blood plasma from the Glaucous gulls were 5.95–44.2 and 0.37–5.61 ng/mL, respectively, whereas those from Baikal seals were 3.57–46.5 and 0.45–2.07 ng/mL, respectively. In both species, rT3 demonstrated low detection rate, while T2 was not detected. Furthermore, cross-array comparison between the HybridSPE®-LC-MS/MS protocol and an established routine radioimmunoassay (RIA) kit-based method was performed for T4 and T3 concentrations from selected Baikal seal plasma samples.

Exploring the Proteomic Alterations from Untreated and Cryoablation and Irradiation Treated Giant Cell Tumors of Bone Using Liquid-Chromatography Tandem Mass Spectrometry.

Giant cell tumors of bone (GCT) are benign tumors that show a locally aggressive nature and affect bones’ architecture. Recently, cryoablation and irradiation treatments have shown promising results in GCT patients with faster recovery and less recurrence and metastasis. Therefore, it became a gold standard surgical treatment for patients. Hence, we have compared GCT-untreated, cryoablation, and irradiation-treated samples to identify protein alterations using high-frequency liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Our label-free quantification analysis revealed a total of 107 proteins (p < 0.01) with 26 up-regulated (<2-folds to 5-fold), and 81 down-regulated (>0.1 to 0.5 folds) proteins were identified from GCT-untreated and treated groups. Based on pathway analysis, most of the identified up-regulated proteins involved in critical metabolic functions associated with tumor proliferation, angiogenesis, and metastasis. On the other hand, the down-regulated proteins involved in glycolysis, tumor microenvironment, and apoptosis. The observed higher expressions of matrix metalloproteinase 9 (MMP9) and TGF-beta in the GCT-untreated group associated with bones’ osteolytic process. Interestingly, both the proteins showed reduced expressions after cryoablation treatment, and contrast expressions identified in the irradiation treated group. Therefore, these expressions were confirmed by immunoblot analysis. In addition to these, several glycolytic enzymes, immune markers, extracellular matrix (ECM), and heat shock proteins showed adverse expressions in the GCT-untreated group were identified with favorable regulations after treatment. Therefore, the identified expression profiles will provide a better picture of treatment efficacy and effect on the molecular environment of GCT.

DNA methylation modification is associated with gonadal differentiation in Monopterus albus

BackgroundBoth testis and ovary can be produced sequentially in an individual with the same genome when sex reversal occurs in the teleost Monopterus albus, and epigenetic modification is supposed to be involved in gonadal differentiation. However, DNA methylation regulation mechanism underlying the gonadal differentiation remains unclear.ResultsHere, we used liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) to simultaneously determine endogenous levels of both 5-methyl-2′-deoxycytidine (m5dC) and 5-hydroxymethyl-2′-deoxycytidine (hm5dC) during gonadal differentiation. Overall DNA methylation level was upregulated from ovary to testis via ovotestis. As a de novo methylase, dnmt3aa expression was also upregulated in the process. Notably, we determined transcription factor Foxa1 for dnmt3aa gene expression. Site-specific mutations and chromatin immunoprecipitation showed that Foxa1 can bind to and activate the dnmt3aa promoter. Furthermore, DNA methylation levels of key genes foxl2 (forkhead box L2) and cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) in regulation of female hormone synthesis were consistently upregulated during gonadal differentiation.ConclusionsThese data suggested that dynamic change of DNA methylation modification is associated with gonadal differentiation.

Valorization of fruit processing by-products: free, esterified, and insoluble bound phytochemical extraction from cherry (Prunus avium) tissues and their biological activities

Fruit processing by-products having limited utilization and no commercial value are generated in high amounts. However, they are rich in phytochemicals with high nutritional value. Therefore, different techniques and applications including extractions, purifications, and fermentations have been evolved for the recovery of these phytochemicals into high value-added products. Similarly, free (F), esterified (E) and insoluble-bound (B) phenolic fractions of sweet cherry processing by-products including stalk, pulp, seed, and leaf were extracted and the potential of these extracts as a source of natural biological compounds were investigated in this study. Total contents of phenolic (TPC), flavonoid (TFC), and hydrolysable tannin compounds (HTC) were analyzed. Individual phenolics were identified and quantified using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). The biological activity of these phytochemicals was determined in terms of antioxidant (DPPH, ABTS, FRAP, and CUPRAC), antidiabetic, and antimicrobial activity. TPC, TFC, and HTC were highly found in F phenolic fractions. Nine phenolic compounds were quantified, most notably in the F phenolic fractions. The enzyme inhibition activity against α-glucosidase (5.88 mg/mL) and α-amylase (0.46 mg/mL) of the E phenolic fractions of seed were significantly higher (p < 0.05) than those of F and B extracts. F fractions of stalk had remarkable antimicrobial activity against selected microorganisms except for Escherichia coli. Two uncorrelated principal components explaining 84.51% of the total variance helped to classify sweet cherry tissues. The results clearly showed that F, E, and B phenolics with their biological properties could be suitable food additives or supplements for enrichment agents in the different formulations.

Hydroethanolic Extract of Lampaya Medicinalis Phil. (Verbenaceae) Decreases Proinflammatory Marker Expression in Palmitic Acid-exposed Macrophages.

Obesity is a major health problem associated with increased comorbidities, which are partially triggered by inflammation. Proinflammatory macrophage infiltration in adipose tissue of individuals with obesity increases chronic inflammation. Obesity is associated with elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which promotes inflammation in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in the folk medicine of Northern Chile to counteract inflammation of rheumatic diseases. Hydroethanolic extract of lampaya (HEL) contains spectrophotometrically defined compounds that may contribute to the observed effect on inflammation. We evaluated the phytochemical composition of HEL by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and liquid chromatography-electrospray ionization- tandem mass spectrometry (LC-ESI-MS/MS). We assessed whether the exposure to HEL affects PA-induced expression of proinflammatory factors in THP-1 macrophages. HPLC-DAD and LC-ESI-MS/MS analyses showed the presence of considerable amounts of flavonoids in HEL. The PA-induced phosphorylation of the inflammatory pathway mediators IKK and NF-κB, as well as the elevated expression and secretion of proinflammatory cytokines (IL-6, TNF-α), were reduced in cells pre-exposed to HEL. These findings give new insights about the effect of HEL reducing IKK/NF-κB proinflammatory pathway, likely explained by the number of flavonoids contained in the extract. More studies would be needed to define the possible role of Lampaya as a preventive approach in subjects with obesity whose circulating PA might contribute to chronic inflammation.