Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines
Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines
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DOI：
10.1002/pmic.200300586
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发表时间：
2003-12-01
期刊：
影响因子：
3.4
通讯作者：
Khoo, KH
中科院分区：
文献类型：
--
作者：
Lee, CL;Hsiao, HH;Khoo, KH
利用互补鸟枪蛋白质组学技术构建了人肝癌HepG 2细胞中最丰富蛋白质的表达图谱。二维液相色谱（LC）-纳米电喷雾电离（ESI）唐德姆质谱（MS/MS），以及一维I-C-基质辅助激光解吸/电离MS/MS进行了评估，并表明，在肽水平引入的额外的分离是不一样有效的简单的预分馏的蛋白质提取物在扩展的范围和总数量的蛋白质鉴定。直接LC-nanoESI MS/MS分析的肽从总溶解的馏分和切下的凝胶带从一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分级不溶的馏分提供了最佳的组合，在有效的建设一个非冗余的细胞图谱。尽管如此，从快速鸟枪蛋白质组学分析的多种变体中编译数据对于增加序列覆盖率和命中置信度仍然很有用，特别是对于主要通过单个或两个肽匹配识别的那些蛋白质。虽然返回的命中分数通常反映了相应蛋白质的丰度，但它不是差异表达的可靠指标。使用另一个密切相关的肝癌Hep 3B作为比较的基础上，16个蛋白质的表达水平超过2倍的差异所定义的斑点强度在二维凝胶电泳分析，其中特别包括热休克蛋白（HSP）和异质性核核糖核蛋白（hnRPN）家族的成员。在Hep 3B中观察到的hnRNPA 2/B1和Hsp 90的较高表达水平导致了对由这两种多功能细胞伴侣共同介导的报道的功能作用的搜索。与端粒酶和端粒结合蛋白在促进其相互作用中所提出的模型一致，获得的数据表明，表达蛋白质组学数据可能与致瘤性Hep 3B中较长的端粒长度相关。这种生物学意义构成了进一步描绘两个蛋白质家族的动态相互作用和修饰的基础，并展示了蛋白质组学和生物学研究如何在假设和模式驱动的研究的生产周期中相互证实。
An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tang dem mass spectrometry (MS/MS) as well as one-dimensional I-C-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research.