• YAKHAK HOEJI
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• v.54 no.5
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• pp.377-386
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• 2010

Single-dimensional (1-D) and two-dimensional (2-D) LC methods were utilized to separate peptides from various sources followed by MS/MS analysis. Two-dimensional ultra-high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than 1-D LC. The most popular 2-D LC approach used today for proteomic research combines strong cation exchange and reversed-phase LC. We have evaluated an alternative mode for 2-D LC of peptides using 2-D RP-RP nano UPLC Q-TOF Mass Spectrometry, employing reversed-phase columns in both separation dimensions. As control experiments, we identified 129 proteins in 1-D LC and 322 proteins in 2-D LC from E. coli extract peptides. Furthermore, we applied this method to rat primary hepatocyte and a total of 170 proteins were identified from 1-D LC, and 527 proteins were identified from all 2-D LC system. The in-depth protein profiling established by this 2-D LC MS/MS from rat primary hepatocyte could be a very useful reference for future applications in regards to drug induced liver toxicity.

• Korean Journal of Food Science and Technology
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• v.31 no.3
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• pp.848-854
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• 1999

Inhibitory compounds of angiotensin converting enzyme (ACE) were separated from Doenjang (traditional Korean fermented soybean paste). Water extracts from Doenjang which showed ACE inhibitory activity were separated with gel permeation chromatography (GPC), in which two fractions with high ACE inhibitory activities were obtained. The first fraction from GPC was further isolated by semi-preparative reverse phase preparative-HPLC (high performance liquid chromatography) and 2-dimensional electrophoresis/thin layer chromatography (TLC). The purified spot had molecular weight of 759 daltons and ninhydrin-positive non-peptide. The second fraction from GPC was also further isolated by semi-preparative reverse phase HPLC and $NH_2-column$ HPLC. One fraction with high ACE inhibitory activity was purified and characterized. Molecular weight of this fraction by LC-MS was 272.34 daltons. The active fraction was identified as Arg-Pro with ACE $IC_{50}$ of $92\;{\mu}M$.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.3
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• pp.275-283
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• 2018

Helicobacter pylori is a causative organism of various gastrointestinal diseases, including chronic gastritis, gastric ulcer, or gastric adenocarcinoma. Pathogenic factors, such as cytotoxin-associated protein A (CagA) and vacuolating cytotoxic protein A (VacA), play a role. This study analyzed qualitatively and quantitatively the effects of zerumbone on the changes in the protein expression levels of various H. pylori proteins, including CagA and VacA. Approximately 200 significant proteins were screened for the H. pylori 60190 (VacA positive / CagA positive; Eastern type) strain, and proteomic analysis was performed on 13 protein molecules that were clinically significant. After two-dimensional electrophoresis (2-DE), $ImageMaster^{TM}$ 2-DE Platinum software was used for quantitative measurements of protein spots. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were used for protein identification. After intensive analysis of the proteins that showed significant changes, a reverse transcription-polymerase chain reaction was performed as required to verify the results. In this study, the significance of zerumbone as a therapeutic agent for H. pylori infection was examined by screening a new pharmacological activity mechanism of zerumbone.

• Journal of Ginseng Research
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• v.45 no.1
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• pp.58-65
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• 2021

Background: Panax ginseng, as one of the most widely used herbal medicines worldwide, has been studied comprehensively in terms of the chemical components and pharmacology. The proteins from ginseng are also of great importance for both nutrition value and the mechanism of secondary metabolites. However, the proteomic studies are less reported in the absence of the genome information. With the completion of ginseng genome sequencing, the proteome profiling has become available for the functional study of ginseng protein components. Methods: We optimized the protein extraction process systematically by using SDS-PAGE and one-dimensional liquid chromatography mass spectrometry. The extracted proteins were then analyzed by two-dimensional chromatography separation and cutting-edge mass spectrometry technique. Results: A total of 2,732 and 3,608 proteins were identified from ginseng root and cauline leaf, respectively, which was the largest data set reported so far. Only around 50% protein overlapped between the cauline leaf and root tissue parts because of the function assignment for plant growing. Further gene ontology and KEGG pathway revealed the distinguish difference between ginseng root and leaf, which accounts for the photosynthesis and metabolic process. With in-deep analysis of functional proteins related to ginsenoside synthesis, we interestingly found the cytochrome P450 and UDP-glycosyltransferase expression extensively in cauline leaf but not in the root, indicating that the post glucoside synthesis of ginsenosides might be carried out when growing and then transported to the root at withering. Conclusion: The systematically proteome analysis of Panax ginseng will provide us comprehensive understanding of ginsenoside synthesis and guidance for artificial cultivation.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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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.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.312-312
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• 2022

Peanut hull as by-product has been discarded during peanut processing. However, peanut hull contains plenty of polyphenols that shows various physiological activities. The objectives of this study were to investigate anti-inflammatory and enzyme inhibitory activities of polyphenols from 'Sinpalkwang' peanut (Arachis hypogaea L.) hull. Compounds were isolated from methanol extracts of peanut hull by preparative-high performance liquid chromatography after identifying and quantifying polyphenols using Ultra performance liquid chromatography (UPLC) and UPLC-Quadrupole time-of-flight-mass spectrometry profiling. The structures of compounds were elucidated by one-dimensional [¹H, ¹³C] nuclear magnetic resonance (NMR) and two-dimensional NMR (correlated spectroscopy, heteronuclear single quantum coherence and heteronuclear multiple bond correlation). Three compounds were identified as 5,7-dihydroxy-4H-chromen-4-one (peak 2), luteolin (peak 4) and eriodictyol (peak 5). Significant differences in inflammatory mediator such as nitric oxide (NO), interleukin-6 (IL-6) and interleukin-1β (IL-lβ) in lipopolysaccharide stimulated Raw 264.7 macrophages and in enzyme (xanthine oxidase [XO] and α-glucosidase [AG]) inhibitory activities were observed between three compounds (p < 0.05). Peak 5 treated Raw 264.7 macrophages showed lower content of NO (16.4 uM), IL-6 (7.0 ng/mL), and IL-1β (60.6 pg/mL) than peak 2 (NO: 28.3 uM, IL-6: 11.3 ng/mL, IL-1β: 66.9 pg/mL) and peak 4 (NO: 24.7 uM, IL-6: 9.3 ng/mL, IL-1β: 62.6 pg/mL). Peak 5 showed higher XO inhibitory activity (84.7%) and higher AG inhibitory activity (52.4%) than peak 2 (XO inhibitory activity: 45.4%, AG inhibitory activity: 21.6%) and peak 4 (XO inhibitory activity: 37.9%, AG inhibitory activity: 37.5%) at concentration of 0.5mg/mL. This study suggests that peanut hull could be a potential source of anti-inflammatory and physiological materials while creating new use of discarded peanut hull as by-products concomitantly.

• Journal of Plant Biotechnology
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• v.37 no.2
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• pp.196-204
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• 2010

With the completion of genome sequencing of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. The recent techniques of proteomics have been advanced quickly so that the high-throughput and systematic analyses of cellular proteins are enabled in combination with bioinformatics tools. Furthermore, the development of proteomic techniques helps to elucidate the functions of proteins under stress or diseased condition, resulting in the discovery of biomarkers responsible for the biological stimuli. Ultimate goal of proteomics orients toward the entire proteome of life, subcellular localization, biochemical activities, and their regulation. Comprehensive analysis strategies of proteomics can be classified as three categories: (i) protein separation by 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification by either Edman sequencing or mass spectrometry (MS), and (iii) quanitation of proteome. Currently MS-based proteomics turns shiftly from qualitative proteome analysis by 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, to quantitative proteome analysis. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. The in vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes, protein-labeling tagging with isotope-coded affinity tag, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope labeled amino acid can be in vivo labeled into live culture cells through metabolic incorporation. MS-based proteomics extends to detect the phosphopeptide mapping of biologically crucial protein known as one of post-translational modification. These complementary proteomic techniques contribute to not only the understanding of basic biological function but also the application to the applied sciences for industry.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.1
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• pp.367-372
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• 2013

Effects of the Epstein-Barr virus (EBV) on cellular protein expression are essential for viral pathogenesis. To characterize the cellular response to EBV infection, differential proteomes of gastric epithelial AGS cells were analyzed with two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and liquid chromatography electrospray/ionization ion trap (LC-ESI-IT) mass spectrometry identification. Mass spectrometry identified 9 altered cellular proteins, including 5 up-regulated and 4 down-regulated proteins after EBV infection. Notably 2-DE analysis revealed that EBV infection induced increased expression of heat shock cognate 71 kDa protein, actin cytoplasmic 1, pyridoxine-5'-phosphate oxidase, caspase 9, and t-complex protein 1 subunit alpha. In addition, EBV infection considerably suppressed those cellular proteins of zinc finger protein 2, cyclin-dependent kinase 2, macrophage-capping protein, and growth/differentiation factor 11. Furthermore, the differential expressional levels of partial proteins (cyclin-dependent kinase 2 and caspase 9) were confirmed by Western blot analysis.Thus, this work effectively provided useful protein-related information to facilitate further investigation of the mechanisms underlying EBV infection and pathogenesis.

• Journal of Microbiology
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• v.45 no.4
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• pp.326-332
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• 2007

In order to understand the functional role of CPRl in Saccharomyces cerevisiae KNU5377 with regard to its multi-tolerance characteristics against high temperatures, inorganic acids, and oxidative stress conditions, whole cellular proteins were analyzed via liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This procedure was followed by two-dimensional (2-D) gel electrophoresis. Under menadione stress conditions, the 23 upregulated proteins were clearly identified only in the wild- type strain of KNU5377. Among the proteins, Sodl1p Tsa1p, Ahp1, Cpr1p, Cpr3, Ssb2p, and Hsp12p were identified as components of antioxidant systems or protein-folding related systems. The CPR1 protein could not be completely detected in the $cpr1{\Delta}$ mutant of KNU5377 and the other upregulated proteins in the wild-type strain evidenced a clear correlation with the results of immunoblot analysis. Moreover, a reduction in growth patterns (about 50%) could be observed in the $cpr1{\Delta}$ mutant, as compared with that of the wild-type strain under mild MD stress conditions. These results indicate that the upregulation of CPR1 may contribute to tolerance against MD as an inducer of oxidative stress.

• ALGAE
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• v.35 no.2
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• pp.177-187
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• 2020

Cyanobacteria have been widely reported to produce a variety of UV-absorbing mycosporine-like amino acids (MAAs). Herein, we reported production of the unusual MAA, mycosporine-glycine-alanine (MGA) in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using a newly developed UHPLC-DAD-MS/HRMS (ultra-high performance liquid chromatography-diode array detection-high resolution tandem mass spectrometry) method. MGA had previously been first identified in a red-algae, but S. torques-reginae strain ITEP-024 is the first cyanobacteria to be reported as an MGA producer. Herein, the chemical structure of MGA is fully elucidated from one-dimensional / two-dimensional nuclear magnetic resonance and HRMS data analyses. MAAs are unusually produced constitutively in S. torques-reginae ITEP-024, and this production was further enhanced following UV-irradiance. It has been proposed that MAA biosynthesis proceeds in cyanobacteria from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate. Annotation of a gene cluster encoded in the genome sequence of S. torques-reginae ITEP-024 supports these gene products could catalyse the biosynthesis of MAAs. However, addition of glyphosate to cultures of S. torques-reginae ITEP-024 abolished constitutive and ultra-violet radiation induced production of MGA, shinorine and porphyra-334. This finding supports involvement of the shikimic acid pathway in the biosynthesis of MAAs by this species.