• Title/Summary/Keyword: MALDI-TOF Mass

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A Systematic Proteome Study of Seed Storage Proteins from Two Soybean Genotypes

  • Cho, Seong-Woo;Kwon, Soo-Jeong;Roy, Swapan Kumar;Kim, Hong-Sig;Lee, Chul-Won;Woo, Sun Hee
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.59 no.3
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    • pp.359-363
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    • 2014
  • Soybean seed is a good source of plant protein in human consumables such as baby formula and protein concentrate. The seeds contain an abundance of storage proteins, namely ${\beta}$-conglycin and glycinin that account for ~ 70-80% of the total seed protein content. Proteome profiling has been proved to be an efficient way that can help us to investigate the seed storage proteins. In the present study, the seeds were removed from the pods and the cotylendonary tissues were separated from the testa for proteome analysis in order to investigate the seed storage proteins. A systematic proteome profiling was conducted through one-dimensional gel electrophoresis followed by MALDI-TOF-TOF mass spectrometry in the seeds (cotyledonary tissue) of soybean genotypes. Two dimensional gels stained with CBB, a total of 10 proteins were identified and analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. A total of ten proteins such as glycinin Gy4 precursor, glycinin G3 precursor, glycinin G1 precursor, glycinin chain A2B1a precursor, glycinin chain A2B1a precursor were identified in our investigation. However, the glycinin subunit may be considered to play important roles in soybean breeding and biochemical characterization. In addition, the improved technique will be useful to dissect the genetic control of glycinin expression in soybean.

Isolation, Purification and Characterization of Antioxidative Bioactive Elastin Peptides from Poultry Skin

  • Nadalian, Mehdi;Kamaruzaman, Nurkhuzaiah;Yusop, Mohd Shakir Mohamad;Babji, Abdul Salam;Yusop, Salma Mohamad
    • Food Science of Animal Resources
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    • v.39 no.6
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    • pp.966-979
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    • 2019
  • Muscle-based by-products are often undervalued although commonly reported having a high amount of natural bioactive peptides. In this study, elastin was isolated from the protein of broiler hen skin while its hydrolysate was prepared using Elastase. Assessment of antioxidative properties of elastin-based hydrolysate (EBH) was based on three different assays; 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical and metal chelating ability. The EBH was purified further using ultrafiltration, gel filtration and Reverse- Phase High-Performance Liquid Chromatography (RP-HPLC). The IC50 of ABTS radical activities for EBH were decreased as EBH further purified using ultrafiltration (EBH III; 0.66 mg/mL)>gel filtration (EB-II; 0.42 mg/mL)>RP-HPLC (EB-II4; 0.12 mg/mL). The sequential identification of the peptide was done by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/ TOF-MS) of the potent fractions obtained from RP-HPLC (EB-II4). The presence of hydrophobic amino acids (Val and Pro) in the peptide sequences could potentially contribute to the high antioxidant activity of EBH. The sequences GAHTGPRKPFKPR, GMPGFDVR and ADASVLPK were identified as antioxidant peptides. In conclusion, the antioxidative potential from poultry skin specifically from elastin is evident and can be explored to be used in many applications such as health and pharmaceutical purposes.

Solid Phase Synthesis of Lysine-exposed Peptide-Polymer Hybrids by Atom Transfer Radical Polymerization (ATRP를 이용한 Lysine 말단기를 가진 펩타이드-고분자 하이브리드 합성)

  • Ha, Eun-Ju;Kim, Mijin;Kim, Jinku;An, Seong Soo A.;Paik, Hyun-Jong
    • Polymer(Korea)
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    • v.38 no.4
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    • pp.550-556
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    • 2014
  • Recently, the peptide(or protein)-polymer hybrid materials (PPs) were sought in many research areas as potential building blocks for assembling nanostructures in selective solvents. In PPs, the facile routes of preparing well-defined peptide-polymer bio-conjugates and their specific activities in various applications are important issues. Our strategy to prepare the peptide-polymer hybrid materials was to combine atom transfer radical polymerization (ATRP) method with solid phase peptide synthesis. The standard solid phase peptide synthesis method was employed to prepare the PYGK (proline-tyrosine-glycine-lysine) peptide. PYGK is an analogue peptide, PFGK (proline-phenylalanine-glycine-lysine), which interacted with plasminogen in fibrinolysis. The peptide and the peptide-initiator were characterized with MALDI-TOF mass spectrometry and $^1H$ NMR spectrometer. The peptide-polymer, pSt-PYGK was characterized by GPC, IR, $^1H$ NMR spectrometer and TLC. Spherical micellar aggregates were determined by TEM and SEM. Current synthesis methodology suggested opportunities to create the well-defined peptide-polymer hybrid materials with specific binding activity.

Identification and Antifungal Susceptibility Profiles of Cyberlindnera fabianii in Korea

  • Park, Ji-Hyun;Oh, Junsang;Sang, Hyunkyu;Shrestha, Bhushan;Lee, Hyeyoung;Koo, Jehyun;Cho, Sung-Il;Choi, Ji Seon;Lee, Min-Ha;Kim, Jayoung;Sung, Gi-Ho
    • Mycobiology
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    • v.47 no.4
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    • pp.449-456
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    • 2019
  • Invasive fungal infections caused by Cyberlindnera fabianii have recently increased. However, biochemical kits such as API 20 C AUX and Vitek-2C have misidentified this species as other Candida spp. such as C. pelliculosa or C. utilis due to no information of Cy. fabianii in yeast database. During our 2016-2017 surveys, eleven isolates of Cy. fabianii were obtained in International St. Mary's Hospital in Korea. Here, we describe its morphological and molecular characteristics and tested its antifungal susceptibility against nine antifungal agents. The sequences of the ITS region and the D1/D2 region of LSU revealed 100% identity with the sequences of Cy. fabianii. In comparison with the results from MALDI-TOF mass spectrometry, we found that Cy. fabianii can be distinguished from other species. In antifungal susceptibility test, voriconazole and echinocandins exhibited good antifungal activities against the majority of Cy. fabianii isolates despite the absence of standard criteria.

Preparation and Properties of Phytosphingosine Ascorbate with Retaining Skin Development Effects (피부 활성을 갖는 Phytosphingosine Ascorbate의 합성)

  • Min, Seok-Kee;Jin, Yong-Hoon;Park, Woo-Jung;Eom, Sang-Yong;Kim, Jong-Heon
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.2
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    • pp.167-172
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    • 2004
  • In the human skin, vitamin C (L -ascorbic acid) that is well known as the activated materials has effects that is skin anti-aging and wrinkle repair by giving impetus to collagen biosynthesis and anti-oxidation, and that is the sun screen, a wound recovering, inhibition melanogenesis and so on. In spite of its effects, vitamin C has the defects of the skin stimulation and easily oxidized instability by water, air, heat and light. For solving their matters, many investigation is advanced and its results are synthesized the various vitamin C derivatives. And yet they have not solved the unstable property of vitamin C and were still insufficient for the comparing with the effect of the pure vitamin C itself. In this study, in order to prepare vitamin C derivative of being improved the stability and to apply vitamin C effect in the skin, we prepared new vitamin C derivative, phytosphingosine ascorbate, by using phytosphingosine, one of sphingolipids, which have a distinguished skin affinity. Phytosphingosine ascorbate can be prepared as the ionic bond between amine group (-NH$_2$) of phytosphingosine and hydroxy group (-OH) of vitamin C by way of the relatively simple reaction. So the structure and properties of the synthesized phytosphingosine ascorbate was confirmed the use of elemental analysis (C 58.3 : H 9.3 : N 2.8 : O 29.5), MALDI TOF-MS (Mw=492.58), Ultraviolet spectra (268.5nm), lH NMR, FT-IR spectra, thermal analysis (m.p=l54$^{\circ}C$), HPLC and so on. And we could confirm the anti-bacterial and anti-oxidation effects. Based on these results, we could confirm to prepare a new material that was expected of both effects of vitamin C and phytosphingosine and that is improved properties of vitamin C.

Proteomic Analyses of Chinese Cabbage(Brassica campestris L. pekinensis) Affected by High Temperature Stresses in Highland Cultivation During Summer in Korea (Proteomics를 이용한 고랭지 배추의 고온장해 해석)

  • Shin, Pyung-Gyun;Hong, Sung-Chang;Chang, An-Cheol;Kim, Sang-Hyo;Lee, Ki-Sang
    • Journal of Life Science
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    • v.17 no.12
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    • pp.1649-1653
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    • 2007
  • High temperature stresses have caused growth inhibition and delayed heading in highland cultivation Chinese cabbage during summer in Korea. We have studied high temperature stress responses in the terms of changes of inorganic components and proteins by proteomic analyses. Insufficiencies of nitrogen and phosphorus have affected growth rate and calcium deficiency has caused blunted heading. Proteins extracted from Brassica seedling grown at the altitude of 600m and 900m in the Mount Jilun were extracted and analysed by 2-dimentional polyacrylamide gel electrophoresis. Profiles of protein expression was then analyzed by 2-dimentional gel analyses. Protein spots showing different expression level were picked using the spot handling workstation and subjected to MALDI-TOF MS. Total 48 protein spots were analyzed by MALDI-TOF MS and 30 proteins spots out of 48 were identified by peptide mass fingerprinting analyses. Fourteen proteins were up-regulated in extracts from the altitude of 900m and they were identified as oxygen-evolving proteins, rubisco activase and ATPase etc. Sixteen proteins were up-regulated in extracts from the altitude of 600m and they were identified as glutathione S-transferase(1, 28kD cold induced- and 24 kD auxin-binding proteins) and salt-stress induced protein etc. These stress-induced proteins were related to the mediated protective mechanism against oxidative damage during various stresses. The results indicated that physiological phenomenon in response to high temperature stresses might be resulted by complex and multiple array of responses with drought, heat, oxidative, salt, and cold by high temperature.

Bacillus anthracis Spores Influence ATP Synthase Activity in Murine Macrophages

  • Seo, Gwi-Moon;Jung, Kyoung-Hwa;Kim, Seong-Joo;Kim, Ji-Cheon;Yoon, Jang-Won;Oh, Kwang-Keun;Lee, Jung-Ho;Chai, Young-Gyu
    • Journal of Microbiology and Biotechnology
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    • v.18 no.4
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    • pp.778-783
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    • 2008
  • Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be down regulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei

  • Yan, Xiang Mei;Kim, Chung-Hyo;Lee, Chul-Kyu;Shin, Jang-Sik;Cho, Il-Hwan;Sohn, Uy-Dong
    • The Korean Journal of Physiology and Pharmacology
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    • v.14 no.2
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    • pp.71-75
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    • 2010
  • To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI- TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration.

Comparative proteomics of the mixotrophic dinoflagellate Prorocentrum micans growing in different trophic modes

  • Shim, Jun-Bo;Klochkova, Tatyana A.;Han, Jong-Won;Kim, Gwang-Hoon;Yoo, Yeong-Du;Jeong, Hae-Jin
    • ALGAE
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    • v.26 no.1
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    • pp.87-96
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    • 2011
  • Protein profiles of a common mixotrophic dinoflagellate, Prorocentrum micans, growing autotrophically and mixotrophically (fed on the cryptophyte Rhodomonas salina) were compared using two-dimensional gel electrophoresis (2-DE) to determine if they vary in different trophic modes. Approximately 2.3% of the detected proteins were differentially expressed in the different trophic modes. Twelve proteins observed only in the mixotrophic condition had lower pI value (<5) than the fifteen proteins observed only in the autotrophic condition (>5). When the internal amino acid sequences of five selected proteins differentially expressed between autotrophic and mixotrophic conditions were analyzed using matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, two proteins that were specifically expressed in the autotrophic condition showed homology to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) and a bacterial catalase. Three mixotrophy-specific proteins showed homology to certain hypothetical proteins from an insect and bacteria. These results suggested the presence of certain gene groups that are switched on and off according to the trophic mode of P. micans.

Enzymatic Production of High Molecular Weight Chitooligosaccharides Using Recombinant Chitosanase from Bacillus thuringiensis BMB171

  • Kang, Lixin;Jiang, Sijing;Ma, Lixin
    • Microbiology and Biotechnology Letters
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    • v.46 no.1
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    • pp.45-50
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    • 2018
  • The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.