Separation and structural analysis of IgG-binding minor proteins in human plasma

人血浆中 IgG 结合次要蛋白的分离和结构分析

• 批准号: 14540561
• 负责人: MANABE Takashi
• 金额: $ 2.3万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14540561/
• 关键词: plasma proteins; two-dimensional electrophoresis; isoelectric focusing; SDS-gel electrophoresis; minor proteins; mass spectrometry; MALDI-TOP MS; ESI-MS; MS; MALDI-TOF MS; 血漿; タンパク質; 電気泳動; 微量; 分離; 構造解析

We have examined the origin of human plasma minor proteins which we found comparing the patterns of two-dimensional gel electrophoresis run under two different conditions of size-separation (second dimension electrophoresis) ; one in the absence of denaturing agents throughout the run and one in the presence of SDS during the size-separation electrophoresis. The minor proteins were analyzed by mass spectrometry, identification of proteins by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flignt mass spectrometer (MALDI-TOF MS) and partial amino acid sequencing by mass-tag method using electrospray ionization tandem mass spectrometer (ESI-MS/MS). Further, we have developed a three-step electrophoresis technique which employ slab-agarose gels in the step of isoelectric focusing and showed that this is useful for the structural analysis of the minor proteins. The comparisons of the patterns obtained by using different conditions of two-dimensional electrophoresis, together with the structural analyses of the proteins on the patterns, will provide insights on the construction processes of functional protein complexes.

我们研究了人类血浆次要蛋白的来源，我们发现比较了两种不同大小分离条件下的双向凝胶电泳图；一种是在整个运行过程中没有变性剂的情况下，另一种是在大小分离电泳过程中存在十二烷基硫酸钠的情况下。利用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)进行蛋白质指纹图谱分析，采用电喷雾串联质谱仪(ESI-MS/MS)进行部分氨基酸序列测定。此外，我们开发了一种三步电泳法，在等电聚焦的步骤中使用平板琼脂糖凝胶，并表明这对次要蛋白质的结构分析是有用的。比较不同条件下的双向电泳图谱，以及图谱上蛋白质的结构分析，将为构建功能蛋白质复合体的过程提供新的思路。

项目成果

島崎洋次, 平加容子, 上杉美夏, 真鍋敬: "Simultaneous Analysis of Esterase and Transferase Activities in Cytosol Proteins from the Bovine Retina"Biochem.Biophys.Acta. 1696. 51-57 (2004)

Yoji Shimazaki、Yoko Hira、Mika Uesugi、Takashi Manabe：“同时分析牛视网膜细胞质蛋白中的酯酶和转移酶活性”Biochem.Biophys.Acta 1696. 51-57 (2004)。

金 亜, 羅国安, 岡 智子, 真鍋 敬: "Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers"Electrophoresis. 23. 3385-3391 (2002)

Jin A，罗国安，Oka Tomoko，Manabe Takashi：“利用毛细管等电聚焦和肽等电点标记估计人血浆蛋白的等电点”电泳23. 3385-3391（2002）。

真鍋 敬: "Analysis of complex protein/polypeptide systems for proteomic studies"J.Chromatogr.B. 787. 29-41 (2003)

Takashi Manabe：“用于蛋白质组学研究的复杂蛋白质/多肽系统的分析”J.Chromatogr.B. 787. 29-41 (2003)

T.Manabe: "Analysis of Complex Protein/Polypeptide Systems for Proteomic Studies"J. Cromatogr. B. 787. 29-41 (2003)

T.Manabe：“蛋白质组学研究中复杂蛋白质/多肽系统的分析”J。

Y.Shimazaki, Y.Sugawara, Y.Ohtsuka, T.Manabe: "Analysis of the Activity and Identification of Enzymes After Separation of Cytosol Proteins in Mouse Liver by Microscale Nondenaturing Two-dimensional Electrophoresis"Proteomics. 3. 2002-2007 (2003)

Y.Shimazaki、Y.Sukawara、Y.Ohtsuka、T.Manabe：“通过微型非变性二维电泳分离小鼠肝脏中的细胞质蛋白后酶的活性分析和鉴定”蛋白质组学。