Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers.
Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers.
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DOI：
10.1186/1477-5956-6-30
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发表时间：
2008-10-24
期刊：
影响因子：
2
通讯作者：
Ngoka LC
中科院分区：
文献类型：
--
作者：
Ngoka LC
实体肿瘤（包括乳腺癌）蛋白质组学的一个重要步骤是有效地提取肿瘤标本中的大部分蛋白质。为此，广泛使用放射免疫沉淀测定（RIPA）缓冲液。RIPA缓冲液的快速和高效的细胞裂解和广泛的蛋白质的良好溶解性进一步增强了其与蛋白酶和磷酸酶抑制剂的相容性，能够最大限度地减少非特异性蛋白质结合，从而降低免疫沉淀的背景，并适用于蛋白质定量。在这项工作中，从乳腺肿瘤中提取蛋白质的RIPA缓冲液提取后留下的不溶性物质进行另一个提取步骤，使用基于尿素的缓冲液。结果表明，RIPA和尿素裂解缓冲液主要根据分子量对乳腺组织蛋白进行裂解。仅溶于尿素缓冲液的蛋白质的平均分子量比RIPA高60%。基因本体论（GO）和有向无环图（DAG）用于映射RIPA和尿素蛋白质组的集体生物和生物物理属性。细胞组分和分子功能注释揭示了缓冲液的蛋白溶解偏好，特别是区室化和功能分布。结果表明，几乎所有的细胞外基质蛋白（ECM）在乳腺肿瘤和匹配的正常组织中被发现，几乎完全，在尿素部分，而它们大多是不溶于RIPA缓冲液。此外，已证明细胞骨架和胞外区蛋白在尿素中比在RIPA中更可溶，而对于核、细胞质和线粒体蛋白，RIPA缓冲液是优选的。细胞外基质蛋白与癌症密切相关，包括其蛋白酶介导的降解和重塑，肿瘤的发展，进展，粘附和转移。因此，如果它们不能被RIPA缓冲液有效地提取，在癌症研究中可能会错过重要的信息。对于实体瘤的蛋白质组学，推荐两步提取过程。首先，应使用RIPA缓冲液提取肿瘤标本中的蛋白质。其次，RIPA不溶性物质应使用本工作中使用的尿素缓冲液进行提取。
An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation. In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA. Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions. It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred. Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research. For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.
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影响因子：
2.9
作者：
GRANIER, F
通讯作者：
GRANIER, F
影响因子：
7
作者：
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影响因子：
2.1
作者：
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通讯作者：
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影响因子：
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通讯作者：
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影响因子：
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作者：
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