Glenn Gary
Ear Institute, University College London, London, United Kingdom.
Methods Enzymol. 2014;536:27-44. doi: 10.1016/B978-0-12-420070-8.00003-9.
The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE.
用于质谱分析和N端测序的蛋白质样品制备是成功鉴定蛋白质的关键步骤。质谱分析是一种非常灵敏的技术，因此，样品必须小心制备，因为它们可能会受到样品污染（例如，由于亚细胞分级分离不完全或多蛋白复合物纯化不完全）、高丰度蛋白质对样品的掩盖以及皮肤或毛发的污染（角蛋白可能是非常常见的干扰因素）。质谱分析样品制备的一个目标是降低样品的复杂性——在此处给出的示例中，线粒体在进行制备型一维SDS-PAGE之前，先通过蔗糖密度梯度沉降进行纯化、溶解和分级分离。验证样品的纯度和完整性很重要，这样你才能对获得的结果有信心。N端测序需要更多的蛋白质，理想情况下，当在SDS-聚丙烯酰胺凝胶上运行时，它应该被纯化到单一条带。此处给出的示例涉及在HEK293细胞中稳定表达一种带标签的蛋白质，然后通过亲和纯化和SDS-PAGE分离该蛋白质。
