A high-throughput approach for subcellular proteome - Identification of rat liver proteins using subcellular fractionation coupled with two-dimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis
A high-throughput approach for subcellular proteome - Identification of rat liver proteins using subcellular fractionation coupled with two-dimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis
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DOI：
10.1074/mcp.m300117-mcp200
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发表时间：
2004-05-01
影响因子：
7
通讯作者：
Zeng, R
中科院分区：
文献类型：
--
作者：
Jiang, XS;Zhou, H;Zeng, R
采用二维液相色谱串联质谱法分析了大鼠肝脏的四种组分（差速离心获得的粗线粒体（CM）和胞质溶胶（C）组分、Nycodenz密度梯度离心获得的纯化线粒体（PM）组分和总肝脏（TL）组分）。共鉴定了564个大鼠蛋白质，并根据其理化特性和功能进行了生物信息学注释。虽然大多数极端碱性核糖体蛋白质中确定的TL馏分，C馏分主要包括中性酶和PM馏分富含碱性蛋白质和蛋白质与电子转移活性或氧结合活性。这些特征在仅在TL、C或PM级分中鉴定的蛋白质中更明显。Swiss-Prot注释和生物信息学预测结果证明C和PM组分分别富集了细胞质或线粒体蛋白。亚细胞分离与二维液相色谱串联质谱联用技术是一种高通量、高灵敏度、高效率的亚细胞蛋白质组学研究方法。使用这样的策略，我们已经构建了最大的蛋白质组数据库迄今大鼠肝脏（564大鼠蛋白质）和细胞质（222大鼠蛋白质）和线粒体组分（227大鼠蛋白质）。此外，在564个鉴定的蛋白质中，使用Swiss-Prot亚细胞位置注释的352个蛋白质作为实际的亚细胞蛋白质组数据集，以评估广泛使用的生物信息学工具，如PSORT，TargetP，TMHMM和GRAVY。
Four fractions from rat liver ( a crude mitochondria ( CM) and cytosol ( C) fraction obtained with differential centrifugation, a purified mitochondrial ( PM) fraction obtained with nycodenz density gradient centrifugation, and a total liver (TL) fraction) were analyzed with two-dimensional liquid chromatography tandem mass spectrometry analysis. A total of 564 rat proteins were identified and were bioinformatically annotated according to their physicochemical characteristics and functions. While most extreme alkaline ribosomal proteins were identified in the TL fraction, the C fraction mainly included neutral enzymes and the PM fraction enriched alkaline proteins and proteins with electron transfer activity or oxygen binding activity. Such characteristics were more apparent in proteins identified only in the TL, C, or PM fraction. The Swiss-Prot annotation and the bioinformatic prediction results proved that the C and PM fractions had enriched cytoplasmic or mitochondrial proteins, respectively. Combination usage of subcellular fractionation with two-dimensional liquid chromatography tandem mass spectrometry was proved to be a high-throughput, sensitive, and effective analytical approach for subcellular proteomics research. Using such a strategy, we have constructed the largest proteome database to date for rat liver ( 564 rat proteins) and its cytosol ( 222 rat proteins) and mitochondrial fractions ( 227 rat proteins). Moreover, the 352 proteins with Swiss-Prot subcellular location annotation in the 564 identified proteins were used as an actual subcellular proteome dataset to evaluate the widely used bioinformatics tools such as PSORT, TargetP, TMHMM, and GRAVY.