Huang Eric L, Piehowski Paul D, Orton Daniel J, Moore Ronald J, Qian Wei-Jun, Casey Cameron P, Sun Xiaofei, Dey Sudhansu K, Burnum-Johnson Kristin E, Smith Richard D
Pacific Northwest National Laboratory (E.L.H., P.D.P., D.J.O., R.J.M., W.-J.Q., C.P.C., K.E.B.-J., R.D.S.), Richland, Washington 99352; and Cincinnati Children's Hospital Medical Center (X.S., S.K.D.), Cincinnati, Ohio 45229.
Endocrinology. 2016 Mar;157(3):1307-14. doi: 10.1210/en.2015-1821. Epub 2016 Jan 8.
Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious approach to achieve in-depth protein coverage and quantitative reproducibility. Biological samples are often severely mass limited and can preclude the application of more robust bulk sample processing workflows. In this study, we present a system that minimizes sample handling by using online immobilized trypsin digestion and solid phase extraction to create a simple, sensitive, robust, and reproducible platform for the analysis of nanogram-size proteomic samples. To demonstrate the effectiveness of our simplified nanoproteomics platform, we used the system to analyze preimplantation blastocysts collected on day 4 of pregnancy by flushing the uterine horns with saline. For each of our three sample groups, blastocysts were pooled from three mice resulting in 22, 22, and 25 blastocysts, respectively. The resulting proteomic data provide novel insight into mouse blastocyst protein expression on day 4 of normal pregnancy because we characterized 348 proteins that were identified in at least two sample groups, including 59 enzymes and blastocyst specific proteins (eg, zona pellucida proteins). This technology represents an important advance in which future studies could perform global proteomic analyses of blastocysts obtained from an individual mouse, thereby enabling researchers to investigate interindividual variation as well as increase the statistical power without increasing animal numbers. This approach is also easily adaptable to other mass-limited sample types.
对纳克级复杂蛋白质样品进行全球蛋白质组分析需要一种严谨的方法，以实现深入的蛋白质覆盖和定量重现性。生物样品通常在质量上受到严重限制，这可能会妨碍应用更强大的大量样品处理工作流程。在本研究中，我们提出了一种系统，该系统通过在线固定化胰蛋白酶消化和固相萃取来尽量减少样品处理，从而创建一个简单、灵敏、稳健且可重现的平台，用于分析纳克级蛋白质组样品。为了证明我们简化的纳米蛋白质组学平台的有效性，我们使用该系统分析了在妊娠第4天通过用盐水冲洗子宫角收集的植入前囊胚。对于我们的三个样品组中的每一组，囊胚分别来自三只小鼠，分别得到22个、22个和25个囊胚。所得的蛋白质组数据为正常妊娠第4天小鼠囊胚的蛋白质表达提供了新的见解，因为我们鉴定了至少在两个样品组中发现的348种蛋白质，包括59种酶和囊胚特异性蛋白质（例如透明带蛋白）。这项技术代表了一项重要进展，未来的研究可以对从单个小鼠获得的囊胚进行全球蛋白质组分析，从而使研究人员能够研究个体间差异，并在不增加动物数量的情况下提高统计效力。这种方法也很容易适用于其他质量受限的样品类型。
