• Mass Spectrometry Letters
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• 제12권3호
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• pp.66-75
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• 2021

Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macrophages or promote the differentiation of secretory cells. The initiation and progression of allergic inflammatory diseases, such as asthma, are dependent on cytokines acting through related receptor complexes. IL-4 and IL-13 are N-glycoproteins. Glycan structures in glycoproteins play important roles in protein folding, protein stability, enzymatic function, inflammation, and cancer development. Therefore, the glycan structure of IL-4 and IL-13 needs to be elucidated in detail for the development of effective therapies. We report the first attempt to characterize the site-specific N-glycosylation of recombinant IL-4 and IL-13 via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The tandem mass spectra of intact N-glycopeptides were identified using the Integrated GlycoProteome Analyzer (I-GPA) platform, which can automatically and rapidly analyze multiple N-glycopeptides, including their glycan composition and amino acid sequences. The recombinant IL-4 and IL-13 were identified with amino acid sequence coverages of 84% and 96%, respectively. For IL-4, 52 glycoforms on one N-glycosylation site were identified and quantified. In IL-13, 232 N-glycopeptides from three N-glycosylation sites were characterized, with the site Asn52 being the most extensively glycosylated (~80%). The complex glycans were the most abundant glycan on IL-4 and IL-13 (~96% and 91%, respectively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.

• 한국식품과학회지
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• 제39권5호
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• pp.488-493
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• 2007

국내유통중인 곡류, 견과류 및 그 가공품 총 25품목, 393건의 시료에 대해 immunoaffinity column 정제방법을 이용하여 총아플라톡신 오염실태를 조사하였으며, 그 결과 곡류 및 곡류가공품 6건, 견과류 및 견과류 가공품 37건에서 아플라톡신 오염이 확인되었으며 오염수준은 아플라톡신 $B_1$으로서 $0.04-2.65{\mu}g/kg$, 총아플라톡신으로서 $0.04-5.51{\mu}g/kg$ 범위로 나타났다. Immunoaffinity column 정제를 거쳐 HPLC-FLD로 분석한 결과 아플라톡신이 검출된 시료에 대해서 LC-MS/MS로 확인하였으며, 그 결과 모두 아플라톡신으로 확인되었다. 본 연구결과에서 나타난 곡류 및 견과류에 대한 아플라톡신 검출빈도 및 오염수준은 국내, 외 연구 결과와 유사하거나 비교적 낮게 나타났으며 국내 아플라톡신 기준 및 미국, CODEX에서 설정된 기준규격 이하로 검출되었다.

• 한국식품과학회지
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• 제43권2호
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• pp.224-229
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• 2011

시중 유통 중인 과자류, 빵, 떡류, 면류 및 선식 등 432건을 immunoaffinity column으로 정제하여 HPLC-FLD로 제랄레논에 대한 오염실태를 조사하였다. 제랄레논의 검량선은 결정계수($R^2$)가 0.999 이상으로 양호한 직선성을 보였고 검출한계 및 정량한계는 각각 2.0, $6.0{\mu}g/kg$, 회수율 80.2-98.4%이었으며 RSD가 0.82-6.40%로 양호한 재현성을 나타내었다. 제랄레논 모니터링 결과, 과자류 중 스낵과자류 66건 중 3건에서 $6.02-11.82{\mu}g/kg$ 검출되었고 비스킷 71건 중 2건에서는 $14.78-17.83{\mu}g/kg$ 검출되었다. 면류, 빵류 및 떡류에서는 제랄레논이 검출되지 않았으며 침출차 14건에서 최고 $53.76{\mu}g/kg$ 검출, 선식 24건 중 16건에서 가장 높은 검출율(66.7%)을 나타내었다. 전분 2건, 시리얼두유 1건에서 제랄레논이 각각 5.55-8.56, $10.26{\mu}g/kg$ 검출되었다. 제랄레논 모니터링 결과는 기준이 설정되어 있는 유럽연합(곡류분말: $75{\mu}g/kg$, 빵, 비스킷, 스낵, 아침식사대용 시리얼류: $50{\mu}g/kg$, 영아 및 유아용 시리얼류: $20{\mu}g/kg$) 기준이하의 수준이었으며 국내외 연구보고와 비교한 결과 낮거나 비슷한 수준으로 검토되었다. 연구결과 곡류가공품에 대한 제랄레논 오염수준은 높지 않았으나 지구 온난화로 기후변화에 민감한 곰팡이독소와 같은 자연독소의 발생 증가가 우려되므로 선제적으로 대응하는 안전관리가 요구된다.

• 한국식품과학회지
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• 제40권3호
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• pp.354-359
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• 2008

국내유통 중인 대두, 붉은콩, 검은콩 녹두 등 6품목의 두류 총 127건을 immunoaffinity column 정제방법 및 HPLC-FLD을 이용하여 제랄레논에 대한 오염실태를 조사하였다. 상관계수($R^2$) 0.999 이상으로 양호한 직선성을 보였고 검출한계 및 정량한계는 각각 2.0 ${\mu}g/kg$, 6.0 ${\mu}g/kg$, 회수율 82.2-98.4%로 나타났다. RSD가 0.82-6.40%로 양호한 재현성을 나타내었다. 모니터링 결과, 대두(백태) 27건 중 1건(3.7%)에서 37.62 ${\mu}g/kg$ 검출되었고 붉은콩(팥) 27건 중 12건 검출되어 44.4%의 검출율을 보였고 그 오염수준은 8.01-38.98 ${\mu}g/kg$이었다. 나머지 시료 검정콩 16건, 녹두 24건, 서리태(속청) 19건, 서목태(약콩) 14건에서는 모두 검출되지 않았다. 따라서 총 시료 127건 중 13건 검출되어 10.2%의 검출율을 보였고 그 오염수준은 8.01-38.98 ${\mu}g/kg$이었다, 검출시료 13건을 LCMS/MS로 확인한 결과 모두 제랄레논임이 확인되었다. 본 연구결과에서 나타난 두류의 검출빈도 및 오염수준은 유럽연합에서 설정된 기준규격 이하의 수준이었으나 곰팡이독소의 생성의 특이성을 고려한다면 지속적이고 광범위하게 오염실태 조사가 되어야 한다고 판단된다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2006년도 추계학술대회
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• pp.65-74
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• 2006

Modem drug discovery requires rapid pharmacokinetic evaluation of chemically diverse compounds for early candidate selection. This demands the development of analytical methods that offer high-throughput of samples. Naturally, liquid chromatography / tandem mass spectrometry (LC-MS/MS) is choice of the analytical method because of its superior sensitivity and selectivity. As a result of the short analysis time(typically 3-5min) by LC-MS/MS, sample preparation has become the rate- determining step in the whole analytical cycle. Consequently tremendous efforts are being made to speed up and automate this step. In a typical automated 96-well SPE(solid-phase extraction) procedure, plasma samples are transferred to the 96-well SPE plate, internal standard and aqueous buffer solutions are added and then vacuum is applied using the robotic liquid handling system. It takes only 20-90 min to process 96 samples by automated SPE and the analyst is physically occupied for only approximately 10 min. Recently, the ultra-high flow rate liquid chromatography (turbulent-flow chromatography)has sparked a huge interest for rapid and direct quantitation of drugs in plasma. There is no sample preparation except for sample aliquotting, internal standard addition and centrifugation. This type of analysis is achieved by using a small diameter column with a large particle size(30-5O ${\mu}$m) and a high flow rate, typically between 3-5 ml/min. Silica-based monolithic HPLC columns contain a novel chromatographic support in which the traditional particulate packing has been replaced with a single, continuous network (monolith) of pcrous silica. The main advantage of such a network is decreased backpressure due to macropores (2 ${\mu}$m) throughout the network. This allows high flow rates, and hence fast analyses that are unattainable with traditional particulate columns. The reduction of particle diameter in HPLC results in increased column efficiency. use of small particles (<2 urn), however, requires p.essu.es beyond the traditional 6,000 psi of conventional pumping devices. Instrumental development in recent years has resulted in pumping devices capable of handling the requirements of columns packed with small particles. The staggered parallel HPLC system consists of four fully independent binary HPLC pumps, a modified auto sampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The process of Bioanalytical Method Validation is required by the FDA to assess and verify the performance of a chronlatographic method prior to its application in sample analysis. The validation should address the selectivity, linearity, accuracy, precision and stability of the method. This presentation will provide all overview of the work required to accomplish a full validation and show how a chromatographic method is suitable for toxirokinetic sample analysis. A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method developed to quantitate drug levels in dog plasma will be used as an example of tile process.

• 한국식품위생안전성학회지
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• 제35권4호
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• pp.326-333
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• 2020

본 연구는 불법적으로 식품에 사용될 수 있는 부정물질 11종에 대한 안전관리 강화를 위해 정량 및 정성 분석이 가능한 HPLC-DAD와 LC-MS/MS를 검증하기 위해 수행되었다. 확립된 시험법은 AOAC 가이드라인에 따라 직선성, 정밀성, 정량한계 및 회수율 등을 통해 유효성을 확인하였다. 본 실험에서 정량한계를 포함하여 검량선을 작성하였고, 모두 0.99 이상의 직선성을 확인하였다. 또한 정확성은 LC (90.0-106%), LC-MS/MS (83.0-114%) 이고, 정밀도는10% 이하로 재현성이 우수하였다. 확립된 시험법은 식품 중 부정물질 안전관리 및 모니터링에 활용될 것으로 사료된다.

• BMB Reports
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• 제43권7호
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• pp.506-511
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• 2010

The levels of salivary cortisol and cortisone in Korean adults were measured for the first time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The salivary cortisol and cortisone were separated within 10 min. The regression coefficients (r) of the calibration curves were greater than 0.999 for the two steroids. The limits of quantitation (LOQ) were 0.2 ng/ml for cortisol and 1 ng/ml for cortisone. The intra-day precisions of the assay were <3.9% and 8.6% for cortisol and cortisone respectively, and the inter-day precisions were <1.9% and 4.3% for cortisol and cortisone, respectively. The salivary cortisone concentrations were approximately 4-9 times higher than those of salivary cortisol during the daytime. Diurnal rhythms, during which the cortisol and cortisone concentrations were higher in the morning than in the afternoon, were also observed. The present assay may be useful for the diagnosis of several adrenal dysfunctions in clinical biochemistry.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.91-95
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• 2005

In the multidimensional protein identification technology of high-throughput proteomics, we use one-dimensional gel electrophoresis and after the separation by two-dimensional liquid chromatography, the sample is analyzed by tandem mass spectrometry. In this study, we have analyzed the Pseudomonas Putida KT2440 protein. From the protein identification, the protein database was combined with its reversed sequence database. From the peptide selection whose error rate is less than 1%, the SEQUEST database search for the tandem mass spectral data identified 2,045 proteins. For each protein, we compared the molecular weight calibrated from 1D-gel band position with the theoretical molecular weight computed from the amino acid sequence, by defining a variable MW$_{corr}$ Since the bacterial proteome is simpler than human proteome considering the complexity and modifications, the proteome analysis result for the Pseudomonas Putida KT2440 could suggest a guideline to build the protocol to analyze human proteome data.

• Safety and Health at Work
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• 제10권2호
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• pp.196-204
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• 2019

Background: Liquid chromatography-tandem mass spectrometry (LC-MSMS) for simultaneous analysis of multiple microbial secondary metabolites (MSMs) is potentially subject to interference by matrix components. Methods: We examined potential matrix effects (MEs) in analyses of 31 MSMs using ultraperformance LC-MSMS. Twenty-one dust aliquots from three buildings (seven aliquots/building) were spiked with seven concentrations of each of the MSMs ($6.2pg/{\mu}l-900pg/{\mu}l$) and then extracted. Another set of 21 aliquots were first extracted and then, the extract was spiked with the same concentrations. We added deepoxy-deoxynivalenol (DOM) to all aliquots as a universal internal standard. Ten microliters of the extract was injected into the ultraperformance LC-MSMS. ME was calculated by subtracting the percentage of the response of analyte in spiked extract to that in neat standard from 100. Spiked extract results were used to create a matrix-matched calibration (MMC) curve for estimating MSM concentration in dust spiked before extraction. Results: Analysis of variance was used to examine effects of compound (MSM), building and concentration on response. MEs (range: 63.4%-99.97%) significantly differed by MSM (p < 0.01) and building (p < 0.05). Mean percent recoveries adjusted with DOM and the MMC method were 246.3% (SD = 226.0) and 86.3% (SD = 70.7), respectively. Conclusion: We found that dust MEs resulted in substantial underestimation in quantifying MSMs and that DOM was not an optimal universal internal standard for the adjustment but that the MMC method resulted in more accurate and precise recovery compared with DOM. More research on adjustment methods for dust MEs in the simultaneous analyses of multiple MSMs using LC-MSMS is warranted.

• Mycobiology
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• 제41권3호
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• pp.149-154
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• 2013

An ${\alpha}$-glucosidase inhibitor was developed from Aspergillus oryzae N159-1, which was screened from traditional fermented Korean foods. The intracellular concentration of the inhibitor reached its highest level when the fungus was cultured in tryptic soy broth medium at $27^{\circ}C$ for five days. The inhibitor was purified using a series of purification steps involving ultrafiltration, Sephadex G-25 gel permeation chromatography, strong cation exchange solid phase extraction, reverse-phase high performance liquid chromatography, and size exclusion chromatography. The final yield of the purification was 1.9%. Results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the purified ${\alpha}$-glucosidase inhibitor was a tri-peptide, Pro-Phe-Pro, with the molecular weight of 360.1 Da. The IC50 value of the peptide against ${\alpha}$-glucosidase activity was 3.1 mg/mL. Using Lineweaver-Burk plot analysis, the inhibition pattern indicated that the inhibitor acts as a mixed type inhibitor.