Atallah Michelle, Flory Mark R, Mallick Parag
Canary Center at Stanford for Cancer Early Detection, Stanford University, 3155 Porter Drive, Palo Alto, CA, 94304, USA.
Methods Mol Biol. 2017;1550:1-10. doi: 10.1007/978-1-4939-6747-6_1.
Proteins play a key role in all aspects of cellular homeostasis. Proteomics, the large-scale study of proteins, provides in-depth data on protein properties, including abundances and post-translational modification states, and as such provides a rich avenue for the investigation of biological and disease processes. While proteomic tools such as mass spectrometry have enabled exquisitely sensitive sample analysis, sample preparation remains a critical unstandardized variable that can have a significant impact on downstream data readouts. Consistency in sample preparation and handling is therefore paramount in the collection and analysis of proteomic data.Here we describe methods for performing protein extraction from cell culture or tissues, digesting the isolated protein into peptides via in-solution enzymatic digest, and peptide cleanup with final preparations for analysis via liquid chromatography-mass spectrometry. These protocols have been optimized and standardized for maximum consistency and maintenance of sample integrity.
蛋白质在细胞稳态的各个方面都起着关键作用。蛋白质组学作为对蛋白质的大规模研究，提供了关于蛋白质特性的深入数据，包括丰度和翻译后修饰状态等，因此为研究生物和疾病过程提供了丰富的途径。虽然诸如质谱等蛋白质组学工具已实现了极其灵敏的样品分析，但样品制备仍然是一个关键的未标准化变量，它会对下游数据读出产生重大影响。因此，在蛋白质组学数据的收集和分析中，样品制备和处理的一致性至关重要。在这里，我们描述了从细胞培养物或组织中提取蛋白质、通过溶液内酶解将分离出的蛋白质消化成肽段以及进行肽段净化并最终制备用于液相色谱 - 质谱分析的方法。这些方案已经过优化和标准化，以实现最大程度的一致性并维持样品完整性。
