• Mass Spectrometry Letters
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• v.7 no.1
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• pp.1-11
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• 2016

Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processes for accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin digestion using an instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in the sample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate the effectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T), and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal tryptic digestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in the number of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed higher numbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis of heat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were compared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestion efficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.

• Plant Biotechnology Reports
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• v.3 no.1
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• pp.67-74
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• 2009

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA ($0.5-8{\mu}M$) or Kn ($0.5-8{\mu}M$) alone or in combination with NAA ($0.5-1.5{\mu}M$). Maximum multiple shoot induction was observed on MS medium supplemented with $4{\mu}M$ BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of $1{\mu}M$ NAA along with $4{\mu}M$ BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA ($5{\mu}M$) and 2,4-D ($2{\mu}M$). The callus was subcultured on MS medium supplemented with BA ($2-15{\mu}M$) or Kn ($2-15{\mu}M$) alone or in combination with NAA ($0.5-2{\mu}M$) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with $10{\mu}M$ BA and $1{\mu}M$ NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA ($1-7{\mu}M$) or IBA ($1-7{\mu}M$). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.

• Mass Spectrometry Letters
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• v.7 no.4
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• pp.106-110
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• 2016

Ginseng, a traditional herbal drug, has been used in Eastern Asia for more than 2000 years. Various ginsenosides, which are the major bioactive components of ginseng products, have been shown to exert numerous beneficial effects on the human body when co-administered with drugs. However, this may give rise to ginsenoside-drug interactions, which is an important research consideration. In this study, acassette assay was performed the inhibitory effects of 12 ginsenosides on seven cytochrome P450 (CYP) isoforms in human liver microsomes (HLMs) using LC-MS/MS to predict the herb-drug interaction. After incubation of the 12 ginsenosides with seven cocktail CYP probes, the generated specific metabolites were quantified by LC-MS/MS to determine their activities. Ginsenoside Rb1 and F2 showed strong selective inhibitory effect on CYP2C9-catalyzed diclofenac 4'-hydroxylation and CYP2B6-catalyzed bupropion hydroxylation, respectively. Ginsenosides Rd showed weak inhibitory effect on the activities of CYP2B6, 2C9, 2C19, 2D6, 3A4, and compound K, while ginsenoside Rg3 showed weak inhibitory effects on CYP2B6. Other ginsenosides, Rc, Rf, Rg1, Rh1, Rf, and Re did not show significant inhibitory effects on the activities of the seven CYPs in HLM. Owing to the poor absorption of ginsenosides after oral administration in vivo, ginsenosides may not have significant side effects caused by interaction with other drugs.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5843-5846
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• 2015

Background: Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. Materials and Methods: A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chisquare test was used to test the differences of PAX1 methylation or HPV infection between groups. Results: The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. Conclusions: The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.

• Analytical Science and Technology
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• v.28 no.1
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• pp.1-7
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• 2015

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco specific nitrosamine found only in tobacco products. The ability to monitor biomarker concentrations is very important in understanding environmental tobacco smoke (ETS). In this study, an efficient and sensitive method for the analysis of NNK in dust was developed and validated using liquid chromatography tandem mass spectrometry. Dust was collected with filter paper soaked in methanol. The standard solution and dust sample were diluted with 100 mM ammonium acetate and extracted using dichloromethane. Our calibration curves ranged from 25 to $10^4pg/mL$. Excellent linearity was obtained with correlation coefficient values between 0.9996 and 1.0000. The limit of detection (LOD) was 5 pg/mL ($S/N{\geq}3$) and the retention time was 10 min. The limit of quantification (LOQ) was 25 pg/mL, and the acceptance criteria was the rate of 98-103% (80-120% at levels up to $3{\times}LOQ$). The coefficient of variations (CV) was 2.8%. Accuracies determined from dust samples spiked with four different levels of NNK racurves ranged that from 25 to 104 pg/mL. Excellent linearity was obtained between 92.1% and 114%. The precision of the method was acceptable (5% of CV). The recovery rates of the whole analytical procedure at low, medium, and high levels were 105.7-116.5% for NNK. The carry-over effects during LC-MS/MS analysis were not observed for NNK. This manuscript summarizes the scientific evidence on the use of markers to measure ETS.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.344-350
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• 2016

Background: Mountain-cultivated ginseng (MCG) and cultivated ginseng (CG) both belong to Panax ginseng and have similar ingredients. However, their pharmacological activities are different due to their significantly different growth environments. Methods: An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS)-based approach was developed to distinguish MCG and CG. Multivariate statistical methods, such as principal component analysis and supervised orthogonal partial-least-squares discrimination analysis were used to select the influential components. Results: Under optimized UPLC-QTOF-MS/MS conditions, 40 ginsenosides in both MCG and CG were unambiguously identified and tentatively assigned. The results showed that the characteristic components of CG and MCG included ginsenoside Ra3/isomer, gypenoside XVII, quinquenoside R1, ginsenoside Ra7, notoginsenoside Fe, ginsenoside Ra2, ginsenoside Rs6/Rs7, malonyl ginsenoside Rc, malonyl ginsenoside Rb1, malonyl ginsenoside Rb2, palmitoleic acid, and ethyl linoleate. The malony ginsenosides are abundant in CG, but higher levels of the minor ginsenosides were detected in MCG. Conclusion: This is the first time that the differences between CG and MCG have been observed systematically at the chemical level. Our results suggested that using the identified characteristic components as chemical markers to identify different ginseng products is effective and viable.

• Bulletin of the Korean Chemical Society
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• v.25 no.10
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• pp.1477-1483
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• 2004

We present the tandem mass spectrometry applications carried out to elucidate the coordination structure of Zn(II) bound lysine ternary complexes, $(Zn+Lys+Lys-H)^+$, which is a good model system to represent a simple (metallo)enzyme-substrate complex (ES). In particular, experimental efforts were focused on revealing the involvement of a lysine side chain ${\varepsilon}$-amino group in the coordination of $Zn^{2+}$ divalent ions. MS/MS fragmentation pattern showed that all the oxygen species within a complex fell off in the form of $H_2O$ in contrast to those of other ternary complexes containing amino acids with simple side chains (4-coordinate geometries, Figure 1a), suggesting that the lysine complexes have different coordination structures from the others. The participation of a lysine basic side chain in the coordination of Zn(II) was experimentally evidenced in MS/MS for $N{\varepsilon}$-Acetyl-L-Lys Zn(II) complexes with acetyl protection groups as well as in MS/MS for the ternary complexes with one $NH_3$ loss, $(Zn+Lys+Lys-NH_3-H)^+$. Detailed structures were predicted using ab initio calculations on $(Zn+Lys+Lys-H)^+$ isomers with 4-, 5-, and 6-coordinate structures. A zwitterionic 4-coordinate complex (Figure 7d) and a 5-coordinate structure with distorted bipyramidal geometry (Figure 7b) are found to be most plausible in terms of energy stability and compatibility with the experimental observations, respectively.

• Analytical Science and Technology
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• v.32 no.5
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• pp.163-172
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• 2019

Methamphetamine (MA) is currently the most abused illicit drug in Korea and its major metabolite is amphetamine (AP). As MA exist as two enantiomers with the different pharmacological properties, it is necessary to determine their respective amounts in a sample. Thus a chiral stationary phase liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of d-MA, l-MA, d-AP, and l-AP in human urine. Urine sample ($200{\mu}L$) was diluted with pure water and purified using solid-phase extraction (SPE) cartridge. A $5-{\mu}L$ aliquot of SPE treated sample solution was injected into LC-MS/MS system. Chiral separation was carried out on the Astec Chirobiotic V2 column with an isocratic elution for each enantiomer. Identification and quantification of enantiomeric MA and AP was performed using multiple reaction monitoring (MRM) detection mode. Linear regression with a $1/x^2$ as the weighting factor was applied to generate a calibration curve. The linear ranges were 25-1000 ng/mL for all compounds. The intra- and inter-day precisions were within 3.6 %, while the intra- and inter-day accuracies ranged from -5.4 % to 11.8 %. The limits of detection were 2.5 ng/mL (d-MA), 3.5 ng/mL (l-MA), 7.5 ng/mL (d-AP), and 7.5 ng/mL (l-AP). Method validation parameters such as selectivity, matrix effect, and stability were evaluated and met acceptance criteria. The applicability of the method was tested by the analysis of genuine forensic urine samples from drug abusers. d-MA is the most common compound found in urine and mainly used by abusers.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.675-683
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• 2018

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

• Bulletin of the Korean Chemical Society
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• v.34 no.7
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• pp.2143-2147
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• 2013

Matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a very effective method for lipid mass fingerprinting. However, MALDI MS suffered from spectral complexities, differential ionization efficiencies, and poor reproducibility when analyzing complex lipid mixtures without prior separation steps. Here, we aimed to find optimal MALDI sample preparation methods which enable selective or class-wide mass fingerprinting of two totally different lipid classes. In order to achieve this, various matrices with additives were tested against the mixture of phosphatidylcholine (PC) and cerebrosides (Cers) which are abundant in animal brain tissues and also of great interests in disease biology. Our results showed that, from complex lipid mixtures, 2,4,6-trihydroxyacetophenone (THAP) with $NaNO_3$ was a useful MALDI matrix for the class-wide fingerprinting of PC and Cers. In contrast, THAP efficiently generated PC-focused profiles and graphene oxide (GO) with $NaNO_3$ provided Cer-only profiles with reduced spectral complexity.