• BMB Reports
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• v.36 no.3
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• pp.299-304
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• 2003

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the co-precipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.401-411
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• 2023

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.1
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• pp.78-85
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• 2019

Membrane proteins are involved in many common diseases, including heart disease and cancer. In various disease states, such as cancer, abnormal signaling pathways that are related to the membrane proteins cause the cells to divide out of control and the expression of membrane proteins can be altered. Membrane proteins have the hydrophobic environment of a lipid bilayer, which makes an analysis of the membrane proteins notoriously difficult. Therefore, this study evaluated the efficacy of two different methods for optimal membrane protein extraction. High-speed centrifuge and reagent-based method with a -/+ filter aided sample preparation (FASP) were compared. As a result, the high-speed centrifuge method is quite effective in analyzing the mitochondrial inner membranes, while the reagent-based method is useful for endoplasmic reticulum membrane analysis. In addition, the function of the membrane proteins extracted from the two methods were analyzed using GeneGo software. GO processes showed that the endoplasmic reticulum-related responses had higher significance in the reagent-based method. An analysis of the process networks showed that one cluster in the high-speed centrifuge method and four clusters in the reagent-based method were visualized. In conclusion, the two methods are useful for the analysis of different subcellular membrane proteins, and are expected to assist in selecting the membrane protein extraction method by considering the target subcellular membrane proteins for study.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.3 no.1
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• pp.99-99
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• 1993

혈액 및 생체시료 중 필수원소 혹은 독극성 원소의 극미량상분 정밀측정과 동위원소비율측정에 널리 사용되는 유도결합플라즈마 질량분석기(ICP-MS)의 기본원리를 소개하고 ICP-SM를 이용한 혈액중 낮은 ppb수준의 Cd, Hg 그리고 Pb의 정밀분석법을 소개한다. 혈액은 많은 양의 유기물을 포함하고 있으므로 digestion bomb에 질산과 과산화수소를 넣어 microwave oven에서 고온고압 상태로 분해시켜 많은 용액을 얻어 이 용액을 플라즈마에 주입시켜 분석한다. 그리고 수온은 tin(II) chloride 용액을 환원제로하여 생성시킨 수은원소증기를 membrane liquid-gas separator를 이용하여 뽑아내어 플라즈마에 주입시켜 낮은 ppt 수준의 검출한계를 얻는다. 또한 높은 정밀도와 정확도와 극미량 원소 측정에 사용되는 동위원소 회석법율 소개하고 실제 혈액분석에의 응용방법을 제시한다.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.384-387
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• 2005

Four new antibiotic peptaibols, named neoatroviridins A (1), B (2), C (3), and D (4), have been isolated from the culture broth of Trichoderma atroviride. Their amino acid sequences were determined mainly by mass spectrometry in combination with NMR studies. The absolute stereochemistry of neoatroviridins was established as L by GC analysis of their acid hydrolysates with derivatization, except for isovaline which was in D. Neoatroviridins, composed of 18 amino acid residues, showed significant membrane-perturbing activity responsible for their antibiotic action, which was comparable to that of alamethicin, a well-known 20-residue peptaibol.

• Journal of the Korean Electrochemical Society
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• v.12 no.2
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• pp.142-147
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• 2009

The influence of graphitization of carbon support on the electrochemical corrosion of carbon and sintering of Pt particles are investigated by measuring $CO_2$ emission at a constant potential of 1.4 V for 30 min using on-line mass spectrometry and cyclic voltammogram. In comparison to commercial Pt/C (from Johnson Matthey), highly graphitized carbon nanofiber (CNF) supported Pt catalyst exhibits lower performance degradation and $CO_2$ emission. As the more carbon corrosion occurred, the more prominent changes were detected in electrochemical characteristics of fuel cell. This indicates that the carbon corrosion affects significantly the fuel cell durability. From the observed results, CNF is considered to be more corrosion resistant material as a catalyst support. However, CNF shows higher aggregation of Pt particles under repeated cyclic voltammetry between 0 and 0.8 V where the carbon corrosion is not initiated.

• Membrane and Water Treatment
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• v.11 no.4
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• pp.257-274
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• 2020

There are many previous review articles are available to summarize either the characterization methods of effluent organic matter (EfOM) or the individual control treatment options. However, there has been no attempt made to compare in parallel the physicochemical treatment options that target the removal of EfOM from biological treatments. This review deals with the recent progress on the characterization of EfOM and the novel technologies developed for EfOM treatment. Based on the publications after 2010, the advantages and the limitations of several popularly used analytical tools are discussed for EfOM characterization, which include UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC), and Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS). It is a recent trend to combine an SEC system with various types of detectors, because it can successfully track the chemical/functional composition of EfOM, which varies across a continuum of different molecular sizes. FT-ICR-MS is the most powerful tool to detect EfOM at molecular levels. However, it is noted that this method has rarely been utilized to understand the changes of EfOM in pre-treatment or post-treatment systems. Although membrane filtration is still the preferred method to treat EfOM before its discharge due to its high separation selectivity, the minimum requirements for additional chemicals, the ease of scaling up, and the continuous operation, recent advances in ion exchange and advanced oxidation processes are greatly noteworthy. Recent progress in the non-membrane technologies, which are based on novel materials, are expected to enhance the removal efficiency of EfOM and even make it feasible to selectively remove undesirable fractions/compounds from bulk EfOM.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• YAKHAK HOEJI
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• v.59 no.3
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• pp.113-119
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• 2015

Protein phosphorylation is one of most important post-translational modifications (PTMs) and plays an important role in regulation of protein function. Here we develop a method for a global identification of phosphopeptides and phosphorylation sites using nano-LC MS/MS. We compared two separation methods, C4 and strong cation ion exchange (SCX). Before phosphopeptides enrichment with $TiO_2$, total proteins from Rat 1 cells have been separated using C4 column or tryptic peptides of proteins from the cells have been separated using SCX column. Finally, we have detected 52 phosphorylation sites on 41 proteins from SCX method and 375 phosphorylation sites on 252 proteins from C4 method, and determined the function and localization of identified phosphoproteins using DAVID software. In particular, we showed new phosphorylation sites from membrane proteins related to various cell signaling mechanisms. This method may contribute to study global signal networks induced by various signals including ligands and drugs.

• Membrane Journal
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• v.15 no.4
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• pp.297-309
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• 2005

As a number of potential endocrine disrupting compounds (EDCs) are released into the environment, recently growing attention has been drawn to them. Therefore sensitive and reliable analytical methods are essential to monitor those compounds. In this study, complementary CC-MS and LC-MS were employed to analyze the endocrine disrupters, and the results of two methods were compared for di(2-ethylhexyl)phthalate (DEHP), benzylbutylphthalate (BBP), pentachlorophenol (PCP), and 4,4'-Isopropylidenediphenol (Bisphenol-A, or BPA). The results indicate that it was possible to lower the detection limits of EDCs by LC-MS. Also, LC-MS enabled to identify the EDCs as almost intact molecules. Furthermore, this study presented a nanofiltration membrane (MWCO 250) and a ultrafiltration membrane (MWCO 1,000) filtration system as methods far removing EDCs from drinking water containing $\gamma$-BHC, p,p'-DDE, BBP, p,p'-DDT, DEHP, PCP, and BPA. Cross-flow type nanofiltrations showed $100\%$ removal of EDCs, and the result implies that MWCO 250 nanofilter was sufficient for treatment of EDCs. The ratio of permeate flux to mass transfer coefficient of nanofiltration, high flux ultrafiltration, and low flux ultrafiltration with ultrapure water were 0.67, 3.4, and 0.44, respectively. It was found that nanofiltration and low flux ultrafiltration were operated at a diffusion dominant condition, and the high flux ultrafiltration was operated at a convection dominant condition. Furthermore, a diffusion dominant process attained reasonable rejection of EDCs. The removal in the ultrafiltration was depending on the molecular weight of an EDC, and the filtration was governed by diffusion-dominant hydrodynamic conditions.