The quantification of ratios between apo to holo types of metal binding protein : a new indicator of the oxidative stress in cells

金属结合蛋白apo与holo类型之间比率的定量：细胞氧化应激的新指标

• 批准号: 11672314
• 负责人: NAKANISHI Toyofumi
• 金额: $ 2.24万
• 依托单位: Osaka Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672314/
• 关键词: NanoESIMS method; Ration of Apo; Holo; Oxidative stress in the body; Cooper-binding protein; Active oxide species; Free cooper ion; Neurodegenerative disease; ホロ型; 遊離銅イオン毒性

We applied nano-electrospray ionization mass spectrometry (NanoESIMS) for the quantification of the rations between apo to holo types of metalloproteins (copper/zinc binding superoxide dismutase:SOD-1, metallothioneine) to clarify the etiology of neurodegenerative diseases such as familial amyotrophic lateral sclerosis. We examined the optimal conditions of ultra-sensitive mass spectrometric quantification of the ratios using metalloproteins standard and purified metalloproteins.1) We examined the condition of pH range of sample solutions, contents of organic solvent and temperature of heated capillary.2) We applied inductively coupled plasma mass spectrometry (ICPMS) instead of nanoESIMS technique.Using the commercial SOD-1 standard, we could not detect dimer, holo type even though we modified some analytical conditions. We give up the construction of the optimal condition of NanoESIMS technique. On the other hand, we applied the ICPMS technique or the de e turn of holo type of SOD-1 and succeeded holo type of the metalloprotem. This technique (ICPMS) was a powerful tool of the quantification of the ratios between apo to holo types of metalloproteins.

We applied nano-electrospray ionization mass spectrometry (NanoESIMS) for the quantification of the rations between apo to holo types of metalloproteins (copper/zinc binding superoxide dismutase:SOD-1, metallothioneine) to clarify the etiology of neurodegenerative diseases such as familial amyotrophic lateral sclerosis.我们检查了使用金属蛋白标准和纯化的金属蛋白对比率进行超敏感的质谱定量的最佳条件。1）我们检查了样品溶液的pH范围的状况，有机溶剂的含量，有机溶剂的含量，加热毛细管的温度含量。即使我们修改了一些分析条件，也无法检测到二聚体类型。我们放弃纳米技术最佳条件的构建。另一方面，我们应用了ICPMS技术或SOD-1的Holo类型的转弯，并成功使用了金属素化剂的Holo类型。该技术（ICPMS）是量化Apo与金属蛋白类型的比率的强大工具。

项目成果

Miyazaki A,Nakanishi T,Kishikawa M,Nakagawa T,Shimizu A,Mawjood AHM,Imai K,Aoki Y & Kikuchi M.: "Compound heterozygosity for b+-thalassemia-31(A->G)and a variant with low oxygan affinity,Hb Sagami[b139(H17)Asn->Thr]"Hemoglobin.. 23・3. 267-271 (1999)

Miyazaki A、Nakanishi T、Kishikawa M、Nakakawa T、Shimizu A、Mawjood AHM、Imai K、Aoki Y 和 Kikuchi M.：“b+-地中海贫血-31（A->G）的复合杂合性和低氧亲和力的变体,Hb 相模[b139(H17)Asn->Thr]“血红蛋白.. 23・3. 267-271 (1999)

Kishikawa M,Nakanishi T,Miyazaki A & Shimizu A.: "Enhanced amyloidogenicity of sulfonated transthyretin in vitro,a hypothetical etiology of senile"Amyloid: Int J Exp Clin Invest. 6・3. 183-186 (1999)

Kishikawa M、Nakanishi T、Miyazaki A 和 Shimizu A.：“磺化转甲状腺素蛋白的体外淀粉样变性增强，老年的假设病因”淀粉样蛋白：Int J Exp Clin Invest 6·3（1999）。

A. Shimizu, T. Nakanishi, M. Kishikawa, A. Miyazaki: "Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis"J. Chromatography B. (in press).

A. Shimizu、T. Nakanishi、M. Kishikawa、A. Miyazaki：“血液和组织中蛋白质变异体和加合物的检测和鉴定：软电离质谱在临床诊断中的应用”J。

T. Hiratsuka, M. Nakazato, T. Minematsu, N. Chiono, T. Nakanishi, A. Shimizu, M. Nakazato, S. Matsukura: "Structural analysis of human β-defensine-1 and its significance in urinary tract infection"Nephron. 85 (1). 34-40 (2000)

T. Hiratsuka、M. Nakazato、T. Minematsu、N. Chiono、T. Nakanishi、A. Shimizu、M. Nakazato、S. Matsukura：“人 β-defensine-1 的结构分析及其在尿路感染中的意义”肾单位。85（1）。

Nakanishi T & Shimizu A.: "Determination of ionization efficiency of glycated and non-glycated peptides from the N-terminal of hemoglobin beta-chain by electrospray ionization mass spectrometry."J.Chromatography (B). 746・1. 83-90 (2000)

Nakanishi T 和 Shimizu A.：“通过电喷雾电离质谱测定血红蛋白 β 链 N 端的糖化和非糖化肽的电离效率。”J.Chromatography (B) 746·1。 (2000)