Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue.
Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue.
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DOI：
10.1002/elps.200800108
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发表时间：
2008-11
期刊：
影响因子：
2.9
通讯作者：
Orton, E. Christopher
中科院分区：
文献类型：
--
作者：
Griffiths, Leigh G.;Choe, Leila;Lee, Kelvin H.;Reardon, Kenneth F.;Orton, E. Christopher
牛心包 (BP) 是一种重要的生物材料，用于生产戊二醛固定心脏瓣膜和组织工程应用。对 BP 进行蛋白质组分析的能力对于一系列研究非常有用，包括植入后免疫排斥的研究。然而，由于 BP 等纤维组织的细胞含量相对较低且细胞外基质丰富，对其进行蛋白质组学分析具有挑战性。研究了多种组织处理、蛋白质提取和分级分离的方法，旨在为水溶性和脂溶性 BP 蛋白质生产高质量的 2-DE 凝胶。用 3-(苄基二甲基铵)-丙磺酸盐提取水溶性蛋白质，然后进行正十二烷基 β-d-麦芽糖苷提取和乙醇沉淀脂溶性蛋白质，提供了 2-DE 凝胶上产量、斑点数和分辨率的最佳组合（方案 E2）。进行 ESI-四极杆/离子阱或 MALDI-TOF/TOF MS 蛋白质鉴定，以确认牛来源和解析蛋白质的适当亚细胞预分级。从水溶性组分中鉴定出二十五种独特的、主要是细胞质的牛蛋白。从脂溶性部分中鉴定出 32 种独特的、主要是膜牛蛋白。这些结果表明，最终方案从这一重要组织中产生了细胞质和膜蛋白的高质量蛋白质组数据。
Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and ;fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl β-d-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.
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影响因子：
4.2
作者：
Bierczynska-Krzysik, Anna;Kang, Sung Ung;Lubec, Gert
通讯作者：
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