Duan Xiaotao, Young Rebeccah, Straubinger Robert M, Page Brian, Cao Jin, Wang Hao, Yu Haoying, Canty John M, Qu Jun
The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York 14260, USA.
J Proteome Res. 2009 Jun;8(6):2838-50. doi: 10.1021/pr900001t.
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 nonionic 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 versus 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. With 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.
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、全面且定量的样品净化和消化程序，以及充分且可重复的色谱分离，都是非常理想的，但仍具有挑战性。然而，最佳方法仍然难以捉摸，尤其是对于富含膜蛋白的蛋白质组，如线粒体。在此，我们描述了一种直接且可重复的样品制备程序，结合高度选择性和灵敏的纳升液相色谱/轨道阱分析，能够实现对组织线粒体可靠且全面的表达谱分析。选择猪心脏的线粒体蛋白质组作为测试系统。使用含有离子和非离子去污剂的强缓冲液完成高效的蛋白质提取。过夜沉淀用于提取物的净化，样品采用优化的两步法、沉淀上消化方法。第一步，在剧烈搅拌下通过4小时胰蛋白酶消化溶解蛋白质沉淀，纳升液相色谱/线性离子阱/电子转移解离显示这会产生大的且未完全裂解的胰蛋白酶肽段。然后将混合物还原、烷基化，并用额外的胰蛋白酶消化成完整的胰蛋白酶肽段。这种溶剂沉淀/沉淀上消化程序比使用用于无标记表达谱分析的常见替代程序（SDS-PAGE/胶内消化）获得了线粒体样品显著更高且更可重复的肽段回收率（87%对54%）。此外，不均匀的肽段损失低于SDS-PAGE/胶内消化。使用具有低死体积、高色谱重现性的纳升液相色谱配置和用于蛋白质鉴定与定量的线性离子阱/轨道阱分析仪，通过5小时梯度充分分离得到的肽段。所开发的方法用于对健康动物与冬眠心肌动物的心肌线粒体蛋白质组进行无标记比较。每个实验组由相对大量的动物组成（n = 10），样品按随机顺序分析以尽量减少定量假阳性。通过这种方法，高置信度地鉴定和定量了904种蛋白质，并将组间显著改变的那些线粒体蛋白质与平行二维差异凝胶电泳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可适用于分析其他组织蛋白质组的进展。
