A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome.
A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome.
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DOI：
10.1021/pr900001t
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发表时间：
2009-06
影响因子：
4.4
通讯作者：
Qu J
中科院分区：
文献类型：
--
作者：
Duan X;Young R;Straubinger RM;Page B;Cao J;Wang H;Yu H;Canty JM;Qu J
对于组织蛋白质组的无标记表达谱分析，高效的蛋白质提取、彻底和定量的样品清理和消化过程以及足够和可重复的层析分离是非常理想的，但仍然具有挑战性。然而，最佳的方法仍然难以捉摸，特别是对于富含膜蛋白的蛋白质组，如线粒体。在这里，我们描述了一种简单且可重复性的样品制备程序，再加上高度选择性和灵敏的Nano-LC/Orbitrap分析，这使得可靠和全面的组织线粒体表达谱成为可能。以猪心脏线粒体蛋白质组为实验系统。高效的蛋白质提取是使用包含离子和非离子洗涤剂的强大缓冲液完成的。用过夜沉淀法对萃取物进行净化，并对样品进行优化的两步颗粒消化方法。在第一步中，在猛烈的搅拌下，经过4h的胰酶消化将蛋白颗粒溶解，纳米LC/LTQ/ETD显示得到大的且不完全被切割的胰蛋白肽。然后将混合物还原、烷基化，并用额外的胰酶将其消化成完整的胰蛋白酶多肽。与目前流行的无标记表达分析的替代方法--SDS-PAGE/凝胶内消化相比，这种溶剂沉淀/颗粒上消化方法获得了显著更高和更可重复性的线粒体制剂的多肽回收率(87%比54%)。此外，不均匀的多肽损失比用SDS-PAGE/凝胶内消化观察到的要少。所得到的多肽通过使用纳米LC构型和LTQ/Orbitrap分析仪进行蛋白质鉴定和定量，通过5h的梯度充分拆分，纳米LC构型具有低空隙体积、高层析重复性。该方法用于健康动物和冬眠动物心肌线粒体蛋白质组的无标记比较。每个实验组由相对较多的动物组成(n=10)，样本按随机顺序进行分析，以最大限度地减少定量假阳性。利用这种方法，904个蛋白质被高置信度地鉴定和定量，并将那些在组间发生显著变化的线粒体蛋白质与平行2D-DGE分析的结果进行比较。这里开发的样品制备和分析策略代表了一种进步，可以适用于分析其他组织蛋白质组。
For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes.
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影响因子：
7
作者：
Cutillas, Pedro R.;Vanhaesebroeck, Bart
通讯作者：
Vanhaesebroeck, Bart
蛋白质组分析前样品制备的蛋白质沉淀方法的比较。
影响因子：
4.1
作者：
Jiang, L;He, L;Fountoulakis, M
通讯作者：
Fountoulakis, M
影响因子：
7
作者：
Ono, Masaya;Shitashige, Miki;Yamada, Tesshi
通讯作者：
Yamada, Tesshi
影响因子：
20.1
作者：
Fallavollita, JA;Malm, BJ;Canty, JM
通讯作者：
Canty, JM
影响因子：
3.4
作者：
Granvogl, Bernhard;Gruber, Patrick;Eichacker, Lutz Andreas
通讯作者：
Eichacker, Lutz Andreas