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Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry 微二维凝胶电泳和基质辅助激光解吸/电离质谱分析了非变性条件下人血浆等电聚焦过程中纤维蛋白原α链的断裂
Pub Date : 2007-09-01 DOI: 10.2198/JELECTROPH.51.27
T. Manabe, Ya Jin, Nao Yamaguchi, T. Sugiyama, Kohei Ikari
The identity of low-molecular-weight and minor protein spots, appeared in 2-DE patterns of human plasma, was examined. They were not obvious in the patterns of “Type-I” 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of “Type II” 2-DE (non-denaturing IEF followed by SDS gel electrophoresis) at pI 5.5-7.5 and apparent mass 8-40 kDa1). The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electrophoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sulfate (AS) fractionation and the 0-35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5-7.5 and apparent mass 8-60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5-7.5 and apparent mass 8-40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C during electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested.
研究了人血浆2-DE模式中出现的低分子量和少量蛋白斑点的一致性。它们在“i型”2-DE(非变性IEF +非变性凝胶电泳)模式中不明显,但在“II型”2-DE(非变性IEF + SDS凝胶电泳)模式中明显存在,在pI 5.5 ~ 7.5,表观质量8 ~ 40 kDa1)。电泳时低温保存(4℃左右)未发现明显斑点,提示其为血浆蛋白水解产物。人血浆经硫酸铵(AS)分馏、0-35%饱和AS馏分透析及ii型2-DE处理时,小斑点更为明显。选取pI 5 ~ 7.5、表观质量8 ~ 60 kDa的2-DE图谱上的116个点,进行MALDI-MS测定,并利用peptide mass fingerprinting (PMF) Mascot和ProFound软件进行质谱分析,确定蛋白质的定位。许多斑点被认为含有纤维蛋白原α链,特别是在pI 5.5 ~ 7.5和表观质量8 ~ 40 kDa的斑点,表明这些斑点是其片段。ms检测到的肽片段的分布表明,分子质量的不均匀性可能是由于α链上多个位点的断裂引起的。在电泳过程中,当人血浆蛋白受到非变性IEF时,必须注意将IEF凝胶的温度保持在4°C左右。ii型2-DE凝胶上没有纤维蛋白原片段斑点,证明血浆蛋白是完整的。指出了微凝胶体系分析完整蛋白混合物的优点。
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引用次数: 6
新規セルロースアセテート膜'セレカ-VSP'の血清タンパク分画検査への評価 : ロット間差および施設間差の多施設共同研究 对新型纤维素醋酸酯膜‘celeka -VSP’的血清蛋白分块检查的评价:多设施合作研究,用于批件间差异和设施间差异
Pub Date : 2007-06-15 DOI: 10.2198/SBK.51.135
弘子 渡邊, 綾 杉浦, 和子 大竹, 皓子 大竹, 深田 比呂子, 康司 堀井, 満 村田, 和代 中村, 秋恵 須郷, 栄治 宮島, 京子 宮﨑, 正芳 米山, 照夫 江上, 卓 渡邊, 芝 紀代子, 真人 前川
In order to substitute SELECA-VSP for SEPARAX-SP, we investigated membrane performance difference among SELECA-VSP lots and among laboratories. We examined three lots of SELECA-VSP, namely T10, T12 and T14. Automated electrophoresis system for protein fraction test used are Olympus AES series in two laboratories and JOKOH CTE series in other two laboratories. The 4 laboratories assayed common serum samples as usual procedure of each laboratory.Among different membrane lots, good correlation between each two lot was observed in the five serum protein fractions. There was statistically significant difference among the membrane lots, however, it might be only due to good reproducibility and statistics. The bias was below clinical permission limit.Among laboratories, good correlation between each two laboratory was observed in the five serum protein fractions. The bias between laboratories is below permission limit, compared with bias detected from external quality assessment program for SEPARAX-SP.In conclusion, because the bias in membrane lots and laboratories can be ignored for laboratory diagnosis of serum protein fraction test, SELECA-VSP will come onto the market to make up for SEPARAX-SP.
为了用SELECA-VSP代替SEPARAX-SP,我们研究了SELECA-VSP批次和实验室之间膜性能的差异。我们检测了三批SELECA-VSP,分别是T10、T12和T14。蛋白组分检测使用的自动电泳系统有两个实验室为Olympus AES系列,另外两个实验室为JOKOH CTE系列。4个实验室按各自实验室的常规程序检测普通血清样本。在不同的膜批次中,血清蛋白的5个组分在每两个批次之间均有良好的相关性。膜组间差异有统计学意义,但这可能只是由于良好的再现性和统计学。偏倚低于临床许可限制。在各实验室中,5个血清蛋白组分之间存在较好的相关性。与SEPARAX-SP外部质量评估程序检测到的偏差相比,实验室之间的偏差低于许可限制。综上所述,由于血清蛋白组分检测在实验室诊断中可以忽略膜批和实验室的偏倚,SELECA-VSP将会上市以弥补SEPARAX-SP的不足。
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引用次数: 0
全自動電気泳動装置(常光CTEシリーズ)による新しいセルロースアセテート膜‘セレカ-VSP’の基本性能評価 基于全自动电泳装置(常光CTE系列)的新型纤维素醋酸膜“celeca -VSP”的基本性能评价
Pub Date : 2007-06-15 DOI: 10.2198/SBK.51.105
和彦 小野寺, 清隆 石原, 秀敏 有園
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引用次数: 0
自動電気泳動装置(AES320)による新規セルロースアセテート膜,セレカ-VSPの臨床検査性能の評価 自动电泳装置(AES320)对新型纤维素醋酸膜celeca -VSP的临床检测性能进行评价
Pub Date : 2007-06-15 DOI: 10.2198/SBK.51.125
弘子 渡邊, 綾 杉浦, 真人 前川
Serum protein fractionation has been performed by electrophoresis on cellulose acetate membrane in clinical laboratory and has been applied to diagnostic procedure for pathological states. In Japan SEPARAX-SP (Fuji film Co.) has been used as the supporting media of cellulose acetate membrane for about twenty years and will finish its production soon. A new cellulose acetate membrane, SELECA-VSP (Toyo Roshi Kaisha Ltd.) has been developed instead of SEPARAX-SP. The SELECA-VSP has almost similar features and function. In this study we evaluated quality and utility of SELECA-VSP by automated electrophoresis system (Olympus AES320). Protein fractionation using SELECA-VSP was not worse than that of SEPARAX-SP. Separation sensitivity was higher in SELECA-VSP. However, irregularly smeared bands were sometimes observed adjacent to monoclonal protein bands. Areas of each fraction are highly correlated in between SEPARAX-SP and SELECA-VSP. The fraction area of α1-globulin was significantly different in SEPARAX-SP and SELECA-VSP, and about 0.01 g / dl higher in SELECA-VSP. After the presence of the difference is recognized, automated classification system for pathological states by protein fractionation could be utilized for routine laboratory testing.In conclusion, the new cellulose acetate membrane, SELECA-VSP will replace SEPARAX-SP as electrophoretic supporting media for serum protein fractionation.
临床实验室已采用醋酸纤维素膜电泳分离血清蛋白,并已应用于病理状态的诊断程序。日本富士胶片公司(SEPARAX-SP)作为醋酸纤维素膜的载体已经使用了近二十年,即将完成生产。一种新的醋酸纤维素膜SELECA-VSP (Toyo Roshi Kaisha Ltd)已被开发出来取代了SEPARAX-SP。SELECA-VSP具有几乎相似的特性和功能。本研究采用自动电泳系统(Olympus AES320)评价SELECA-VSP的质量和效用。selca - vsp对蛋白质的分离效果不差于SEPARAX-SP。SELECA-VSP的分离灵敏度较高。然而,在单克隆蛋白条带附近,有时会观察到不规则的涂抹条带。每个分数的面积在SEPARAX-SP和selca - vsp之间高度相关。α1-球蛋白在SEPARAX-SP和SELECA-VSP中的分数面积差异显著,且SELECA-VSP中α1-球蛋白的分数面积高出0.01 g / dl。识别差异存在后,通过蛋白质分离的病理状态自动分类系统可用于常规实验室检测。综上所述,新型醋酸纤维素膜SELECA-VSP将取代SEPARAX-SP作为血清蛋白分离的电泳载体。
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引用次数: 1
Comparison of solubilization and preparation procedures for membrane-enriched proteome analysis 富膜蛋白质组分析的增溶和制备方法比较
Pub Date : 2007-06-01 DOI: 10.2198/JELECTROPH.51.15
Y. Tsujimoto, N. Taguchi, Kiyoo Hirooka, Naohiro Tomari, Akiko Okami, T. Nishikawa, Y. Nakazawa, Kunihiko Watanabe, H. Matsui, Yoshihiro Yamamoto
Proteome analysis of membrane proteins by two-dimentional electrophoresis (2-DE) is still insufficient due to the problem of membrane proteins solubilization. Additionally, nucleic acids and lipids are known to interfere with the profile on 2-DE, and removal of these substances is often required. In this study, three reagents from Bio-X Inc. were used to remove these interfering substances from protein. Prior to loading, samples were treated with the reagents for 30-60 min at 30°C. Pretreatment with the reagents led to high-resolution separations of proteins. Here, we describe how sample pretreatment using the reagents improved proteomic maps of proteins extracted from Escherichia coli and Saccharomyces cerevisiae. To remove lipids enhanced the solubility and separation of membrane proteins from cells.
由于膜蛋白的溶解问题,利用二维电泳(2-DE)对膜蛋白进行蛋白质组学分析仍然不足。此外,已知核酸和脂质会干扰2-DE的轮廓,通常需要去除这些物质。在本研究中,使用Bio-X公司的三种试剂去除蛋白质中的这些干扰物质。在上样前,用试剂在30℃下处理样品30-60 min。用这些试剂进行预处理可以实现高分辨率的蛋白质分离。在这里,我们描述了样品预处理如何使用试剂改善从大肠杆菌和酿酒酵母中提取的蛋白质的蛋白质组学图。去脂增强了膜蛋白与细胞的溶解性和分离性。
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引用次数: 2
タンデム四重極リニアイオントラップを用いた微量タンパク質の検出及び翻訳後修飾研究への戦略4000 Q TRAP®システム 4000 Q TRAP®系统,用于检测和翻译后修饰的微量蛋白质,使用串联四极磁离子捕捉
Pub Date : 2007-03-21 DOI: 10.2198/SBK.51.23
文彦 土屋
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引用次数: 1
MS Spectrometer application in life sciences : Developments of sample preparation and differential analysis as a data analysis platform 质谱仪在生命科学中的应用:作为数据分析平台的样品制备和差分分析的发展
Pub Date : 2007-03-21 DOI: 10.2198/SBK.51.7
Masato Ushiyama, Y. Ishizuka, J. Hirano
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引用次数: 0
The protein expression profile of cynomolgus monkey embryonic stem cells in two-dimensional gel electrophoresis: a successful identification of multiple proteins using human databases 食蟹猴胚胎干细胞二维凝胶电泳蛋白表达谱:利用人类数据库成功鉴定多种蛋白
Pub Date : 2007-03-01 DOI: 10.2198/JELECTROPH.51.1
Masako Nakahara, K. Saeki, Yoshiko Yogiashi, A. Kimura, A. Horiuchi, Naoko Nakamura, Asako Yoneda, Koichi Saeki, Satoko Matsuyama, Megumi Nakamura, T. Toda, Yasushi Kondo, Y. Kaburagi, A. Yuo
Global gene and protein expression analyses have had great impacts on scientific progresses in this new era of bioinformatics. Although studies using murine and human materials can fully exploit the large volume of their databases, there are quite a few inconveniences for an investigation on non-human primate materials due to still insufficient data collections. Here we examined the availability of human databases for the protein identification process using the two-dimensional electrophoresis-based proteomic study in cynomolgus monkey embryonic stem (ES) cells. Querying public human protein databases, we successfully identified multiple protein spots via mass spectrometric analysis using MALDI-TOF apparatus. The results of the protein identification were confirmed by western blotting using polyclonal antibodies raised against human epitopes. Interestingly, the results of western blotting further identified the existence of previously unreported multiple isoforms of common proteins including glycolytic pathway enzymes. Thus, combined analyses of the mass spectrometry querying the Homo sapience databases and the western blotting using polyclonal antibodies is highly effective in determining protein expressions in monkey cells. Our success in obtaining a draft protein expression profile of cynomolgus monkey ES cells will contribute to the promotion of non-human primate ES cell researches.
在生物信息学的新时代,全球基因和蛋白质表达分析对科学进步产生了重大影响。虽然使用小鼠和人类材料的研究可以充分利用其庞大的数据库,但由于数据收集仍然不足,对非人灵长类动物材料的调查存在不少不便。在这里,我们使用基于二维电泳的蛋白质组学研究,检测了人类数据库中蛋白质鉴定过程的可用性,用于食蟹猴胚胎干(ES)细胞。通过查询公共人类蛋白质数据库,利用MALDI-TOF仪器进行质谱分析,成功鉴定出多个蛋白质位点。蛋白鉴定结果经western blotting(免疫印迹法)证实,抗体为抗人表位的多克隆抗体。有趣的是,western blotting的结果进一步确定了包括糖酵解途径酶在内的常见蛋白质的多种亚型的存在。因此,将查询智人数据库的质谱分析与使用多克隆抗体的western blotting相结合,在测定猴细胞中的蛋白表达方面是非常有效的。我们成功获得食蟹猴胚胎干细胞的蛋白表达谱草图,将有助于促进非人类灵长类动物胚胎干细胞的研究。
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引用次数: 2
Carbonic anhydrase II was detected in urine of a patient with osteosarcoma during high-dose methotrexate and leucovorin rescue therapy 在高剂量甲氨蝶呤和亚叶酸素抢救治疗期间,在骨肉瘤患者的尿液中检测到碳酸酐酶II
Pub Date : 2007-03-01 DOI: 10.2198/JELECTROPH.51.9
Toyoji Sato, T. Kaneko, N. Ishikawa, Takako Toyama, S. Enomoto, T. Ōhashi, Yukio Shima, A. Kimura, H. Hatano, T. Uchiyama, Hiroyuki Segawa, Hiroto Kobayashi, T. Morita
Using a 5–20% (linear gradient) polyacrylamide slab gel electrophoresis, we studied the urinary protein collected from a 14-year-old girl with osteosarcoma during high-dose methotrexate (MTX) and leucovorin rescue therapy. A distinct protein band of approximately 30 kDa (P-30 protein) was observed in addition to albumin. We have never before encountered a 30 kDa urinary protein. Therefore, we first studied the P-30 protein by peptide mass fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We obtained the amino acid sequence “YDPSLKPLSVSYDQATSLR”, which is the same as that of amino acids (40–58) of human carbonic anhydrase II (CAII). Then, we confirmed this protein by Western blotting using anti-human CAII antibody. This enzyme distributes widely in the human bodies, in erythrocytes, kidney, and pancreas. We considered that this CAII originated from the kidney in association with high-dose MTX and leucovorin rescue therapy.
采用5-20%(线性梯度)聚丙烯酰胺平板凝胶电泳,研究了一名14岁骨肉瘤女孩在大剂量甲氨蝶呤(MTX)和亚叶酸素抢救治疗期间采集的尿蛋白。除白蛋白外,还观察到约30 kDa的明显蛋白带(P-30蛋白)。我们以前从未遇到过30kda的尿蛋白。因此,我们首先利用多肽质量指纹图谱和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对P-30蛋白进行了研究。我们得到的氨基酸序列为“YDPSLKPLSVSYDQATSLR”,与人类碳酸酐酶II (CAII)的氨基酸序列(40-58)相同。然后用抗人CAII抗体进行Western blotting证实该蛋白。这种酶广泛分布于人体内的红细胞、肾脏和胰腺中。我们认为这种CAII起源于肾脏,与大剂量甲氨蝶呤和亚叶酸素抢救治疗有关。
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引用次数: 0
セパラックス-SP(富士フイルム製)からセレカ-VSP(東洋濾紙製)への移行 从separax -SP(富士胶卷制造)到sereca -VSP(东洋滤纸制造)的转变
Pub Date : 2007-01-01 DOI: 10.2198/sbk.51.101
英孝 岡田, 俊憲 新城
The stable supply of the cellulose acetate membrane (SEPARAX-SP) for electrophoresis support media became difficult with production device deterioration. Therefore, Toyo Roshi Kaisha, Ltd. was transferred production technology from Fuji Co. and tried to develop a new membrane with similar performance. At the beginning physical properties of the membrane differed slightly possibly due to the difference of production device; however, recently a new membrane with similar performance to SEPARAX-SP has been completed and named SELECA-VSP.Physical properties, such as tearing strength and elasticity, electrophoretic properties of SELECA-VSP were similar to those of SEPARAX-SP. By scanning electron microscope, SELECA-VSP membrane surface and cross section showed similar image and uniformly pored.To investigate a practical use of SELECA-VSP in clinical laboratory, a working group was organized by Japanese Electrophoresis Society and has examined basic and clinical performance of SELECA-VSP. Because the results have been revealed almost good performance, we go on developing for placing SELECA-VSP on the market.
随着生产设备的老化,用于电泳支撑介质的醋酸纤维素膜(SEPARAX-SP)的稳定供应变得困难。因此,东洋Roshi Kaisha株式会社从富士株式会社引进生产技术,试图开发出具有类似性能的新型膜。由于生产设备的不同,膜的初始物理性能略有不同;然而,最近一种与SEPARAX-SP性能相似的新膜已经完成,并被命名为SELECA-VSP。SELECA-VSP的撕裂强度、弹性、电泳等物理性能与SEPARAX-SP相似。通过扫描电镜观察,SELECA-VSP膜表面和横截面图像相似,孔隙均匀。为了研究SELECA-VSP在临床实验室的实际应用,日本电泳学会组织了一个工作组,对SELECA-VSP的基础和临床性能进行了研究。由于结果显示出几乎良好的性能,我们继续开发将SELECA-VSP投放市场。
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引用次数: 0
期刊
Journal of capillary electrophoresis
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