Proteomic analysis of "Oriental Beauty" oolong tea leaves with different degrees of leafhopper infestation.

RATIONALE: Oriental Beauty, a type of oolong tea native to Taiwan, is highly prized by connoisseurs for its unique fruity aroma and sweet taste. Leaves of Oriental Beauty vary in appearance, aroma, and taste, depending on the degree of tea green leafhopper (Jacobiasca formosana) infestation. In this study, the aim is to investigate the differential expression of proteins in leaves with low, medium, and high degrees of leafhopper infestation. METHODS: Proteomic techniques 2DE (two-dimensional electrophoresis) and nanoscale liquid chromatography/tandem mass spectrometry (LC/MS/MS) were used to investigate the differential expression of proteins in tea leaves with different degrees of leafhopper infestation. RESULTS: A total of 89 proteins were found to exhibit significant differences in expression. In a gene ontology analysis, most of these proteins participated in biosynthesis, carbohydrate metabolism, transport, responses to stress, and amino acid metabolism. CONCLUSIONS: These results indicated that the unique aroma and taste of the leaves might be influenced by their protein expression profiles, as well as related factors such as defensive responses to tea green leafhopper saliva.

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

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

Rapid determination of bioactive compounds in the different organs of Salvia Miltiorrhiza by UPLC-MS/MS.

Salvia miltiorrhiza has been widely used in Asia for medicinal purposes for >1000 years due to the high levels of bioactive constituents it contains. In this study, a simple and rapid ultrasound-assisted liquid extraction (5 min) was applied for the extraction of these bioactive constituents. The extracts were analyzed by using rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with simultaneous positive and negative electrospray ionization in a single analytical run. Eight analytes were separated within 2.2 min during 6 min of run time with UHPLC-MS/MS operated in multiple reaction monitoring (MRM) mode. The concentration of salvianolic acids and tanshinones in the different organs of different varieties of Salvia miltiorrhiza ranged from 6.4 to 382.1 mg/g and 0.03 to 31.7 mg/g, respectively. To the best of our knowledge, this is the first report to characterize the tanshinone compounds found in the flower and stem/leaf of Salvia miltiorrhiza by UHPLC-MS/MS.

A rapid, simple, and high-throughput UPLC-MS/MS method for simultaneous determination of bioactive constituents in Salvia miltiorrhiza with positive/negative ionization switching.

The dried root of Danshen (Salvia miltiorrhiza) is a Chinese medicine that has been used in traditional treatments for more than 2000 years in Asia. It has also been used to treat cardiovascular diseases for decades. The goal of this study was to develop a new, fast, and simple ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytical method for the analysis of phenolic acids and tanshinones in Danshen. The samples were separated on a Kinetex F5 column (2.1 x 100 mm, 1.7 µm, pentafluorophenyl) using 78% acetonitrile with 0.1% formic acid as the mobile phase with isocratic elution while the flow rate was set at 0.45 mL/min. The analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) in polarity switching mode. The linearity values of the calibration curves of eight compounds were in the range from 3 to 3200 ng/mL (r2 > 0.99). Method validation was performed in terms of linearity, the relative standard deviations (RSDs) of the intra-day and inter-day, and it was found that precisions were within 15% and the accuracy ranged from 93% to 118%. The recovery was more than 87%. The limit of detection (LOD) and limit of quantification (LOQ) values of the eight analytes varied between 0.023 to 0.75 ng/mL and 0.375 to 1.5 ng/mL, respectively. This approach has the shortest analysis time for the separation of phenolic acids and tanshinones in Danshen using a UPLC core-shell column with F5 stationary phase. Meanwhile, this robust and high-throughput method not only has a short run time (2 min) but also reduce the consumption of organic solvents.

Comparative Proteomic Analysis of Rat Bronchoalveolar Lavage Fluid after Exposure to Zinc Oxide Nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in consumer products and industrial applications. As a result of all these uses, this has raised concerns regarding their potential toxicity. We previously found that candidate markers of idiopathic pulmonary fibrosis and lung cancer were significantly up-regulated in rat bronchoalveolar lavage fluid (BALF) following exposure to ZnO NPs by using a liquid chromatography (LC)-based proteomic approach. To achieve comprehensive protein identification analysis, we conducted the two-dimensional gel electrophosis (2-DE)-based proteomic workflow to analyze the differences in BALF proteins from rats that had been exposed to a high dose of 35 nm ZnO NPs. A total of 31 differentially expressed protein spots were excised from the gels and analyzed by nanoLC-tandem mass spectrometry (MS/MS). Gene ontology (GO) annotation of these proteins showed that most of the differentially expressed proteins were involved in response to stimulus and inflammatory response processes. Moreover, pulmonary surfactant-associated protein D and gelsolin, biomarkers of idiopathic pulmonary fibrosis, were significantly up-regulated in rat BALF after ZnO NPs exposure (2.42- and 2.84-fold, respectively). The results obtained from this present study could provide a complementary consequence with our previous study and contribute to a better understanding of the molecular mechanisms involved in ZnO NP-induced lung disorders.

2-DE combined with two-layer feature selection accurately establishes the origin of oolong tea.

Taiwan is known for its high quality oolong tea. Because of high consumer demand, some tea manufactures mix lower quality leaves with genuine Taiwan oolong tea in order to increase profits. Robust scientific methods are, therefore, needed to verify the origin and quality of tea leaves. In this study, we investigated whether two-dimensional gel electrophoresis (2-DE) and nanoscale liquid chromatography/tandem mass spectroscopy (nano-LC/MS/MS) coupled with a two-layer feature selection mechanism comprising information gain attribute evaluation (IGAE) and support vector machine feature selection (SVM-FS) are useful in identifying characteristic proteins that can be used as markers of the original source of oolong tea. Samples in this study included oolong tea leaves from 23 different sources. We found that our method had an accuracy of 95.5% in correctly identifying the origin of the leaves. Overall, our method is a novel approach for determining the origin of oolong tea leaves.

Graphene flakes enhance the detection of TiO2-enriched catechins by SALDI-MS after microwave-assisted enrichment.

In this study, we describe a novel method for the rapid detection of catechins in tea leaves using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The method involves incubating catechins in a mixture of titanium dioxide (TiO2) nanoparticles and graphene flakes (GF) and then subjecting the mixture to microwave irradiation to facilitate the enrichment of analytes. In this approach, TiO2 nanoparticles serve as probes to enrich catechins while GF serve to increase the desorption/ionization efficiency. The use of the TiO2-GF mixture as matrix provides a number of advantages over TiO2 nanoparticles alone, including higher desorption/ionization efficiency, enhanced analyte enrichment and reduced run time (less 10min). We demonstrate that the addition of GF to nanoparticles of TiO2 improves the detection of catechins by SALDI-MS. At a signal-to-noise ratio of 3, the limits of detection were 0.04pmole for (+)-catechin (C), 0.17pmole for (-)-epigallocatechin (EGC), 0.35pmole for (-)-epicatechin gallate (ECG), and 0.50pmole for (-)-epigallocatechin gallate (EGCG). This method was further applied to determine the catechin content in several tea samples and the results showed good shot-to-shot and sample-to-sample reproducibility (less than 10% and 13%, respectively) as well as good quantitative linearity for the four analytes (R(2)>0.995). The precision calculated as percent relative standard derivation (%RSD), ranged from 3.6% to 12.4%. Overall, we show that this method is a sensitive, simple and high-throughput technique for the detection of catechins in tea.

Changes in protein expression in rat bronchoalveolar lavage fluid after exposure to zinc oxide nanoparticles: an iTRAQ proteomic approach.

RATIONALE: Zinc oxide nanoparticles (ZnO NPs) are widely used in consumer products and various biomedical fields. As a result, humans are frequently exposed to these NPs. However, there is a lack of information about the proteins that are expressed in the airway in response to exposure to ZnO NPs. METHODS: Bronchoalveolar lavage fluid (BALF) from Sprague-Dawley (SD) rats that had been exposed to high-dose 35 nm ZnO NPs (N = 6) and filtered air (N = 4) was collected and then labeled with isobaric tags for relative and absolute quantitation (iTRAQ). The differentially expressed proteins were identified by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) and further classified by Gene Ontology (GO) annotation. RESULTS: A total of 46 proteins displayed significant changes after exposure. GO annotation of these differentially expressed proteins indicated that exposure to ZnO NPs mainly affected immune and inflammatory processes. Furthermore, S100A8 and S100A9, candidate markers of idiopathic pulmonary fibrosis and lung cancer, were significantly up-regulated (2.78- and 2.87-fold, respectively) following exposure. CONCLUSIONS: Our data are consistent with recent study results that exposure to ZnO NPs induces lung inflammation. These data contribute to a better understanding of how exposure to ZnO NPs leads to lung damage through the functional classification of these proteins.