Two-dimensional Liquid Chromatography-tandem Mass Research Articles

A Proteomic Study on Seed Germination of Nitraria roborowskii Kom.

Owing to the dormancy of the seeds of Nitraria roborowskii Kom., which results in a low germination rate in nature, germination takes a long time, and natural regeneration is difficult. Therefore, there is a need to study the molecular mechanism by which the seeds of N. roborowskii release dormancy. In this study, the differentially expressed proteins of N. roborowskii seeds before and after dormancy and germination were quantitatively and qualitatively analyzed via two-dimensional high-performance liquid chromatography tandem mass spectrometry (LC–MS) and TMTTM. Differentially expressed proteins from dormant and germinated seeds were characterized and enriched via bioinformatics to determine the functions and pathways of the differentially expressed proteins. The results revealed that seed dormancy was regulated by multiple metabolic pathways, including protein synthesis, nutrient utilization and phytohormone signal transduction pathways. A comparison of the dormant and germinated N. roborowskii seed samples revealed 1082 differentially expressed proteins with FC ≥ 1.5 and p values ≤ 0.05, among which 191 proteins were upregulated and 891 proteins were downregulated in the seeds of the germinated group, and proteins more closely related to the key genes of the germinated N. roborowskii seeds were involved in the activity of D-threo-aldose 1-dehydrogenase. Four proteins (WD40, cystatin, AMP binding protein, and helicase) were involved in the positive regulation of seed germination. The release of N. roborowskii seed dormancy is a complex biological process involving cell differentiation, formation, cellular transport, signaling and resistance, etc. The interactions of multiple metabolic pathways, such as carbon fixation, glycolysis/gluconeogenesis, the pentose phosphate pathway, endoplasmic reticulum protein processing and pyruvic acid metabolism in photosynthetic organisms, constitute a complex regulatory mechanism for dormancy release.

Ultrasensitive quantification of trace amines based on N-phosphorylation labeling chip 2D LC-QQQ/MS

Trace amines (TAs) are metabolically related to catecholamine and associated with cancer and neurological disorders. Comprehensive measurement of TAs is essential for understanding pathological processes and providing proper drug intervention. However, the trace amounts and chemical instability of TAs challenge quantification. Here, diisopropyl phosphite coupled with chip two-dimensional (2D) liquid chromatography tandem triple-quadrupole mass spectrometry (LC-QQQ/MS) was developed to simultaneously determine TAs and associated metabolites. The results showed that the sensitivities of TAs increased up to 5520 times compared with those using nonderivatized LC-QQQ/MS. This sensitive method was utilized to investigate their alterations in hepatoma cells after treatment with sorafenib. The significantly altered TAs and associated metabolites suggested that phenylalanine and tyrosine metabolic pathways were related to sorafenib treatment in Hep3B cells. This sensitive method has great potential to elucidate the mechanism and diagnose diseases considering that an increasing number of physiological functions of TAs have been discovered in recent decades.

A sensitive, heart-cutting, two-dimensional liquid chromatography–tandem mass spectrometry method for the determination of mometasone furoate in human plasma: Application for a bioequivalence study in nasal spray formulations

AbstractWe developed and validated a sensitive, heart-cutting, two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC‒MS/MS) method to determine the concentration of mometasone furoate in human plasma after nasal spray administration. Isotopically labeled mometasone furoate-13C,d6 was used as an internal standard (IS). Plasma samples were prepared using a solid-phase extraction (SPE) method. With this 2D-LC strategy, the analytes were trapped in the first dimension (1D) column, and only judiciously selected portions of the 1D effluent were transferred to the second dimension (2D) column for further separation to obtain high-resolution information. MS/MS quantification was performed in positive ionization mode via multiple-reaction monitoring (MRM). This analytical method was fully validated according to related regulatory guidance, and the results showed that the method is robust and sensitive enough for pharmacokinetic investigation of mometasone furoate with satisfactory linearity from 0.25 to 30 pg mL−1. This method was successfully applied to a bioequivalence (BE) study of mometasone furoate aqueous nasal sprays in healthy volunteers.

A fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry and its application in food-derived protein hydrolysates

Food-derived bioactive peptides have many outstanding features like high safety, easy absorption, etc. However, explorations of the peptides are suffering from the limited knowledge of sample composition and low efficiency of separation techniques. In this work, a fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS) was designed and constructed in-house. For chromatographic system optimization, the effects of column pairs and fraction transfer volumes on separation performance were studied. The pair of Protein BEH SEC and HSS T3 columns was found of high orthogonality. The peak capacity detected by the optimized 2DLC reached 1165 (for corn protein hydrolysates), indicating high resolving power. Moreover, the number of peptides identified from corn, soybean and casein protein hydrolysates reached as high as 8330, 8925 and 7215, respectively, demonstrating the high potential of the system. This would help reveal the peptide composition and facilitate the research on exploring bioactive peptides from food-derived protein hydrolysates.

2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Quantitative Proteomics-Based Analysis Reveals Molecular Mechanisms of Chilling Tolerance in Grafted Cotton Seedlings

Proteome analysis of grafted cotton exposed to low-temperature stress can provide insights into the molecular mechanistic of chilling tolerance in plants. In this study, grafted and non-grafted cotton plants were exposed to chilling stress (10 °C/5 °C) for 7 d. After the stress, rootstock and scion samples were labeled by 8-plex iTRAQ (isobaric Tags for Relative and Absolute Quantification), followed by two-dimensional liquid chromatography separation and tandem mass spectrometry identification. In total, 68 differential proteins were identified that were induced by low-temperature stress and grafting, and these proteins regulate physiological functioning. Under low-temperature stress, in the cotton seedlings, the proteins responded to the MAPK signaling pathway and calcium signaling pathway enhanced, the metabolisms of carbohydrate, lipid, nucleotide, and amino acid had a tendency to intensify, the proteins related to protein folding and degradation were activated, along with the system of antioxidant enzymes to offset cellular oxidative damage. In contrast, chilling stress reduced oxidative phosphorylation, photosynthesis, and carbon fixation. These data indicated that the physiological changes in cotton seedlings comprise a complex biological process, and the ability of plants to resist this stress can be improved after grafting onto a vigorous rootstock, although this was not obvious in the young plants. Further studies of low-temperature stress and/or graft-related differences in proteins could lead to the identification of new genes associated with chilling tolerance in plants. These data provide the basis for further studies on the molecular mechanism of chilling tolerance and the relationship of grafting and chilling tolerance in cotton.

DIA Comparative Proteomic Analysis of Retro-oil Fluid and Vitreous Fluid From Retinal Detachment Patients.

Objectives: There have been reports of unexplained visual loss following intra-ocular silicone oil (SiO) tamponade in retinal detachment patients, yet the underlying mechanism is unknown. The aim of this study was to investigate the mechanisms behind retinal toxicity following intra-ocular SiO tamponade in retinal detachment patients. Methods and Results: Vitreous fluid samples were acquired from 27 patients (27 eyes). Twelve eyes for data-independent acquisition (DIA) were divided into four groups: pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RD group), SiO removal after successful retinal reattachment (SO group), cataract surgery after successful retinal reattachment with sterilized air tamponade (FA group), and PPV for epiretinal membrane (ERM group). The remaining 15 eyes were used for enzyme-linked immunosorbent assay analysis. DIA was combined with two-dimensional liquid chromatography–tandem mass spectrometry to find expression changes in the proteome of vitreous. Mean number mass spectra, statistically differentially expressed proteins, gene ontology (GO), pathway representations, and protein interactions were analyzed. GO analysis showed that the protein categories of synapse organization, cell adhesion, and regulation of cell migration in the SO group were differentially expressed compared to the control or FA groups (p < 0.05). Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, lysosome and cell adhesion were found to be significantly enriched in the SO group compared to the FA and control groups (p < 0.05). Cadherin 2, transferrin, and lysosome function may partially contribute to silicone oil-related vision loss. Conclusion: Vision loss-inducing novel molecular signatures and pathways that may be associated with SiO toxicity were identified. Transferrin may be a potential visual outcome biomarker for SiO tamponade.

Proteome profiling of human placenta reveals developmental stage-dependent alterations in protein signature

IntroductionPlacenta is a complex organ that plays a significant role in the maintenance of pregnancy health. It is a dynamic organ that undergoes dramatic changes in growth and development at different stages of gestation. In the first-trimester, the conceptus develops in a low oxygen environment that favors organogenesis in the embryo and cell proliferation and angiogenesis in the placenta; later in pregnancy, higher oxygen concentration is required to support the rapid growth of the fetus. This oxygen transition, which appears unique to the human placenta, must be finely tuned through successive rounds of protein signature alterations. This study compares placental proteome in normal first-trimester (FT) and term human placentas (TP).MethodsNormal human first-trimester and term placental samples were collected and differentially expressed proteins were identified using two-dimensional liquid chromatography-tandem mass spectrometry.ResultsDespite the overall similarities, 120 proteins were differently expressed in first and term placentas. Out of these, 72 were up-regulated and 48 were down-regulated in the first when compared with the full term placentas. Twenty out of 120 differently expressed proteins were sequenced, among them seven showed increased (GRP78, PDIA3, ENOA, ECH1, PRDX4, ERP29, ECHM), eleven decreased (TRFE, ALBU, K2C1, ACTG, CSH2, PRDX2, FABP5, HBG1, FABP4, K2C8, K1C9) expression in first-trimester compared to the full-term placentas and two proteins exclusively expressed in first-trimester placentas (MESD, MYDGF).ConclusionAccording to Reactome and PANTHER softwares, these proteins were mostly involved in response to chemical stimulus and stress, regulation of biological quality, programmed cell death, hemostatic and catabolic processes, protein folding, cellular oxidant detoxification, coagulation and retina homeostasis. Elucidation of alteration in protein signature during placental development would provide researchers with a better understanding of the critical biological processes of placentogenesis and delineate proteins involved in regulation of placental function during development.

Two-dimensional liquid chromatography - mass spectrometry as an effective tool for assessing a wide range of pharmaceuticals and biomarkers in wastewater-based epidemiology studies

The biomarkers used in wastewater-based epidemiology studies have a wide range of physicochemical properties, making simultaneous analysis extremely challenging or even impossible. So far, the majority of analytical procedures employed in this field have focused on specific classes or a limited number of analytes. The heart-cutting two-dimensional liquid chromatography tandem mass spectrometry technique (2D-LC–MS/MS) has enabled the incorporation of compounds with significantly different polarities in a single analytical method.In this study, a 2D-LC–MS/MS method has been developed and optimized for the simultaneous analysis of various biomarkers in wastewater, providing relevant information about lifestyle habits (the usage of alcohol, tobacco, and caffeine), health and well-being (pharmaceuticals), and a population size biomarker (5-HIAA) that is useful for correct calculation of human population during wastewater-based epidemiology studies. A single-laboratory validation procedure showed satisfactory analytical performance, with the mean trueness for most compounds falling in the range of 80÷110 % and the mean repeatability being less than 20 %. The applicability of the method was evaluated by testing 28 wastewater samples collected at different wastewater treatment plants in Latvia. The obtained results revealed the occurrence of 44 out of 62 selected biomarkers, with the lowest quantified concentration at 1.1 ng L−1 in the case of xylometazoline and up to 148 μg L−1 in the case of metformin and 156 μg L−1 for caffeine.

Proteomic profiling of clinical and environmental strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous bacterium, which is able to change its physiological characteristics in response to different habitats. Environmental strains are presumably less pathogenic than clinical strains and whether or not the clinical strains originate from the environment or through inter-host transmission remains poorly understood. To minimize the risk of infection, a better understanding of proteomic profiling of P. aeruginosa is necessary for elucidating the correlation between environmental and clinical strains. Based on antimicrobial susceptibility and patterns of virulence, we selected 12 clinical and environmental strains: (i) environmental, (ii) multidrug resistant (MDR) clinical and (iii) susceptible clinical strains. Whole-cell protein was extracted from each strain and subjected to two-dimensional differential gel electrophoresis (2-D DIGE) and liquid chromatography tandem mass spectrometry quadrupole time-of-flight (LC-MS QTOF). All 12 strains were clustered into 3 distinct groups based on their variance in protein expression. A total of 526 matched spots were detected and four differentially expressed protein spots (p < 0.05) were identified and all differential spots were downregulated in MDR strain J3. Upregulation of chitin binding and BON domain proteins was present in the environmental and some MDR strains, whereas the clinical strains exhibited distinct proteomic profiles with increased expression of serine protein kinase and arginine/ornithine transport ATP-binding proteins. Significant difference in expression was observed between susceptible clinical and MDR strains, as well as susceptible clinical and environmental strains. Transition from an environmental saprophyte to a clinical strain could alter its physiological characteristics to further increase its adaptation.

Quantitative proteomic profiling of Cervicovaginal fluid from pregnant women with term and preterm birth

BackgroundPreterm birth (PTB) is one of major causes of perinatal mortality and neonatal morbidity, but knowledge of its complex etiology is still limited. Here we present cervicovaginal fluid (CVF) protein profiles of pregnant women who subsequently delivered at spontaneous preterm or term, aiming to identify differentially expressed CVF proteins in PTB and term birth.MethodsThe CVF proteome of women who sequentially delivered at preterm and term was analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS). We compared the CVF proteome of PTB (n = 5) and control subjects (term birth, n = 7) using pooled control CVF (term birth, n = 20) as spike-in standard.ResultsWe identified 1294 CVF proteins, of which 605 were newly identified proteins. Of 990 proteins quantified in both PTB and term birth, 52 proteins were significantly up/down-regulated in PTB compared to term birth. The differentially expressed proteins were functionally associated to immune response, endopeptidase inhibitors and structural constituent of cytoskeleton. Finally, we confirm the down-regulation of SERPINB7 (a serine-type protease inhibitor) in PTB compared to control by Western blot.ConclusionsTaken together, our study provide quantitative CVF proteome profiles of pregnant women who ultimately delivered at preterm and term. These promising results could help to improve the understanding of PTB etiology and to discover biomarkers for asymptomatic PTB.

Evidence of free radical generation from the interaction of polyethylene glycol with PVC medical tubing

The combination of polyethylene glycol (PEG) and polyvinyl chloride (PVC) medical tubing was previously demonstrated to degrade an active pharmaceutical ingredient (API), a phenomenon proposed to occur by free radical mechanisms. This study tests the hypothesis that dehydrochlorinated PVC at the tubing surface increases the oxidative potential of PEG autooxidation via radical propagation. The functional group composition at the surfaces of intact, autoclaved, or force-degraded medical grade PVC tubings was assessed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The content of double bonds in PVC was correlated with the extent of API degradation in the PEG–PVC system, with the repeated autoclaving cycle treatments yielding the most reactive tubing. After PEG exposure, new functional groups on the surface of PVC were observed, indicating the participation of PVC in the oxidation reactions. The PEG–PVC system was further probed by the fluorinated spin-trap reagent FDMPO, where trapped adducts were analyzed by 19F NMR, revealing the presence of three radical species. Trapped adducts were then analyzed by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS), which revealed the presence of free chlorine atoms and/or hypochlorous acid and a PEG alkoxy radical. Chemical mechanisms describing the interaction between dehydrochlorinated PVC and PEG are proposed to explain the presence of free radicals and the functional group changes in the PVC surface.

Identification of Candidate Biomarkers for EGFR-T790M Drug-resistant Gene Mutation in Advanced Lung Adenocarcinoma

背景与目的奥希替尼（Osimertinib）是美国食品和药物管理局（Food and Drug Administration, FDA）批准用于携带表皮生长因子受体（epidermal growth factor receptor, EGFR）-T790M突变的晚期非小细胞肺癌（non-small cell lung cancer, NSCLC）患者的药物，用药前需行EGFR-T790M检测。不少患者因进展病灶隐匿或体弱无法进行组织活检错过Osimertinib治疗，本研究希望能从血清中筛查出预测EGFR-T790M耐药突变相关蛋白，为临床用药提供帮助。本研究旨在探索EGFR-T790M耐药基因相关蛋白，为临床用药提供帮助。方法本研究纳入口服易瑞沙晚期肺腺癌患者36例，在疾病进展后行组织活检，使用ARMS方法检测出EGFR-T790M突变组患者18例，非EGFR-T790M突变组18例。收集耐药患者血清，采用同位素标记相对和绝对定量标记结合二维液相色谱串联质谱蛋白组学技术筛选与EGFR-T790M耐药相关蛋白。结果筛出17种差异性蛋白，与EGFR-T790M基因突变相关上调蛋白6种，下调蛋白11种，主要参与31种生物过程，7种细胞组分，26种分子功能；反应途径中共鉴定出12种富集途径，其中富集指数最高的是凝血级联途径。结论发现与EGFR-T790M耐药相关蛋白共17种，参与凝血级联途径蛋白有望成为预测EGFR-T790M耐药突变相关的生物标志物。

Effect of transient scrotal hyperthermia on human sperm: an iTRAQ-based proteomic analysis

BackgroundThrough this prospective study, we aimed to explore the change of molecular modification after the transient scrotal hyperthermia on human sperm.MethodsTen healthy subjects selected with strict screening criteria underwent testicular warming in a 43 °C water bath for 30 min a day for 10 consecutive days. Semen samples were collected 2 weeks before the first heat treatment and 6 weeks after the first heat treatment. Proteins from the samples were labeled with isobaric tags for relative and absolute quantitation and analyzed by two-dimensional liquid chromatography–tandem mass spectrometry.ResultsIn contrast to the control, of the 3446 proteins identified, 61 proteins were deregulated: 28 were up-regulated and 33 were down-regulated. Approximately 95% of the differentially expressed proteins were found to participate in spermatogenesis, fertilization, or other aspects of reproduction. In particular, the expression of sperm motility and energy metabolism-related proteins AKAP4, SPESP1, ODF1, ODF2, GAPDHS, and ACTRT2, validated by western blotting of the proteins obtained from human and mouse samples, tended to be reduced under scrotal hyperthermia.ConclusionsThe results indicated that the proteins AKAP4, ODF1, ODF2, GAPDHS, SPESP1, and ACTRT2, play an important role in the heat-induced reversible reduction in sperm concentration and motility and have the potential to be the biomarkers and clinical targets for scrotal heat treatment induced male infertility.

Comparative Proteomics and Secretomics Revealed Virulence and Antibiotic Resistance-Associated Factors in Vibrio parahaemolyticus Recovered From Commonly Consumed Aquatic Products.

Vibrio parahaemolyticus is a seafoodborne pathogen that can cause severe gastroenteritis and septicemia diseases in humans and even death. The emergence of multidrug-resistant V. parahaemolyticus leads to difficulties and rising costs of medical treatment. The bacterium of environmental origins containing no major virulence genes (tdh and trh) has been reported to be associated with infectious diarrhea disease as well. Identification of risk factors in V. parahaemolyticus is imperative for assuming food safety. In this study, we obtained secretomic and proteomic profiles of V. parahaemolyticus isolated from 12 species of commonly consumed aquatic products and identified candidate protein spots by using two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry techniques. A total of 11 common and 28 differential extracellular proteins were found from distinct secretomic profiles, including eight virulence-associated proteins: outer membrane channel TolC, maltoporin, elongation factor Tu, enolase, transaldolase, flagellin C, polar flagellin B/D, and superoxide dismutase, as well as five antimicrobial and/or heavy metal resistance-associated ABC transporter proteins. Comparison of proteomic profiles derived from the 12 V. parahaemolyticus isolates also revealed five intracellular virulence-related proteins, including aldehyde-alcohol dehydrogenase, outer membrane protein A, alkyl hydroperoxide reductase C, phosphoenolpyruvate-protein phosphotransferase, and phosphoglycerate kinase. Additionally, our data indicated that aquatic product matrices significantly altered proteomic profiles of the V. parahaemolyticus isolates with a number of differentially expressed proteins identified. The results in this study meet the increasing need for novel diagnosis candidates of the leading seafoodborne pathogen worldwide.

Protective effects of cardiac resynchronization therapy in a canine model with experimental heart failure by improving mitochondrial function: a mitochondrial proteomics study.

Cardiac resynchronization therapy (CRT) is well acknowledged as an effective treatment for dyssynchronous heart failure. However, the molecular mechanism is unclear to date. Mitochondrial dysfunction and impaired energetic metabolism are two important mechanisms that lead to heart failure. Therefore, we aim to screen the changes of mitochondria-associated proteins and signaling pathways involved in heart failure and CRT treatment. A total of 24 beagle dogs were randomly assigned into control (CON), heart failure (HF), or CRT group. Myocardial mitochondria from the free wall of left ventricle was extracted for isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography tandem mass spectrometry analysis (2DLC-MS/MS). A total of 2190 proteins were identified, among which 234 proteins were differentially expressed in HF compared with CON group, 151 proteins were differentially expressed in CRT compared with HF group. A total of 192 of the 234 differentially expressed proteins in HF group were changed oppositely by CRT treatment, and 128 of the 151 CRT-induced differentially expressed proteins showed opposite trend of expression to HF/CON. Gene Ontology analysis of the 128 proteins revealed that 16 were localized in mitochondria, 17 were associated with calcium signaling, and 7 could be secreted extracellularly for cell-to-cell signaling. Calpain-1 (CAPN1), which is localized to mitochondria and related to calcium signaling, was upregulated in HF and downregulated after CRT treatment. CRT treatment also improved mitochondrial morphology and function and reduced collagen areas of both interstitial and perivascular fibrosis. CRT treatment significantly improved cardiac function, reduced myocardial fibrosis, and enhanced mitochondrial function in the failing heart through CAPN1 downregulation.

Validation of a universal and highly sensitive two-dimensional liquid chromatography–tandem mass spectrometry methodology for the quantification of pyrazinamide, ethambutol, protionamide, and clofazimine in different biological matrices

A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3–111.4%) and accuracies (92.1–109.3%), together with high precision values (0.5–13.8%), were verified in all matrices. All standard curves showed favorable linearities with r2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague–Dawley rats after oral administration of the drug combination.

Determination of mycotoxins in beer by multi heart-cutting two-dimensional liquid chromatography tandem mass spectrometry method

Beer is one the most consumed alcoholic beverage in the world and its contamination with mycotoxins is of public health concern. This study reports a fast and automated analytical procedure based on a multi-heart-cutting two-dimensional liquid chromatography tandem mass spectrometry method using electrospray ionization for the determination of seven mycotoxins (aflatoxins B1, B2, G2 and G1, ochratoxin A, fumonisins B1 and B2) in beers. The developed method was based on the heart-cutting 2D- HPLC technique in which only the specific portions of the first dimension, in the retention time of analytes, were transferred into the second dimension for the further separation and successive determination. The method uses two different chromatographic columns; in the first dimension, 50 μL of sample was injected on first column, and mycotoxins elution regions were collected in a loop and transferred into the second column for the separation of analytes. Each column operated in gradient elution mode in order to eliminate interfering compounds and improve separation and peak shape. After the optimization, the method has been validated according to EU regulation and finally applied for the analysis of forty beer samples collected from Italian supermarkets. Among all mycotoxins studied, fumonisins B1 was the most widely distributed in analysed beers (>21%) in the range from 0.6 to 12.3 ng mL−1. The automated methodology developed was able to determine accurately and simultaneously seven mycotoxins in beer. This provided a significant reduction of sample handle and, consequently of analysis time.

Serum proteins TGFBI, PCSK9, and CCL14 are potential biomarkers for different traditional Chinese medicine syndromes of multidrug-resistant tuberculosis.

Patients with multidrug-resistant tuberculosis (MDR-TB) tend to have a long course of anti-TB treatment and severe side effects. Traditional Chinese Medicine (TCM) has a synergistic effect in attenuation of MDR-TB. However, the lack of objective biological standards to classify and diagnose MDR-TB TCM syndromes could result in less effective TCM treatment. Therefore, in this study, we identified differentially expressed proteins (DEPs) in serum of individuals with MDR-TB TCM syndromes by applying isobaric tags for relative and absolute quantification coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) method and bioinformatics analysis. The functional analysis of DEPs was also performed. Additionally, DEPs among three different TCM syndromes of MDR-TB were validated by enzyme-linked immunosorbent assay (ELISA). Finally, a receiver operating characteristic (ROC) curve was performed to estimate the diagnostic ability of DEPs. A total of 71 DEPs were identified in the three different MDR-TB TCM syndrome groups such as the pulmonary Yin deficiency (PYD) syndrome group, the Hyperactivity of Fire due to Yin deficiency (HFYD) syndrome group, and the deficiency of Qi and Yin (DQY) syndrome group. The results showed that the expression level of transforming growth factor-beta-induced protein ig-h3 (TGFBI) was lower in the PYD syndrome group (p = .002), the proprotein convertase subtilisin/kexin type 9 (PCSK9) was overexpressed in the HFYD syndrome group (p < .0001), and the C-C motif chemokine ligand 14 (CCL14) expression level was reduced in the DQY syndrome group (p = .004). Our study demonstrated that serum TGFBI, PCSK9, and CCL14 may serve as potential novel biomarkers for PYD syndrome, HFYD syndrome and DQY syndrome of MDR-TB, respectively. The study provides a biological basis for MDR-TB TCM syndromes classification and can be of great significance for the treatment of different TCM syndromes.

Comparative proteomic analysis of resistant and susceptible potato cultivars during Synchytrium endobioticum infestation.

We report the first comparative study of protein expression profiles in tuber sprouts between Katahdin-derived potato cultivars resistant and susceptible to Synchytrium endobioticum. Synchytrium endobioticum causes wart disease in potato (Solanum tuberosum L.) and is considered as the most important quarantine pathogen in almost all countries where potatoes are grown. We performed a comparative analysis of differentially expressed proteins in the tuber sprouts of potato cultivars differing in resistance to pathotype 1(D1) of S. endobioticum using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches. Bulks prepared from two resistant (Calrose and Humalda) and three susceptible (Sebago, Seneca and Wauseon) potato cultivars were studied. When protein profiles were compared between mock- and S. endobioticum-inoculated sprouts, 35 and 63 protein spots, indicating qualitative or quantitative differences, were detected in the resistant and susceptible cultivars, respectively. In turn, 24 proteins associated with resistance to S. endobioticum were revealed by comparison of the resistant and susceptible bulks. These proteins were changed in a constitutive or induced manner and were grouped into four categories: stress and defence, cell structure, protein turnover, and metabolism. Among the 13 proteins classified into the stress and defence group, seven proteins were related to heat-shock proteins (HSPs)/chaperone factors. In addition, four proteins, S-adenosyl-L-homocysteine hydrolase-like, superoxide dismutase [Mn], inactive patatin-3-Kuras 1 and patatin-15, were induced in the resistant bulk; whereas two proteins, patatin-01 and nucleoredoxin 1, showed significant differences in expression between the S. endobioticum-inoculated resistant and susceptible bulks. The detection of such a large number of S. endobioticum-mediated proteins representing the HSP70, HSP60 and HSP20 families suggests their significant role in restricting wart disease in potato tubers.