Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.
Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Nat Protoc. 2017 Nov;12(11):2391-2410. doi: 10.1038/nprot.2017.100. Epub 2017 Oct 26.
Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.
外部干扰或内部信号引起的蛋白质结构变化可以深刻影响蛋白质的活性，从而调节细胞生理。有许多生物物理方法可用于探测蛋白质结构变化，但这些方法不适用于生物提取物中的整个蛋白质组。有限蛋白酶解-质谱联用（LiP-MS）是一种新开发的蛋白质组学方法，可直接在蛋白质组范围内其复杂的生物学背景下识别蛋白质结构变化。在感兴趣的扰动之后，蛋白质组提取物首先在天然条件下用非特异性蛋白酶进行双蛋白酶消化步骤，然后在变性条件下用序列特异性蛋白酶胰蛋白酶进行完全消化。这种顺序处理生成适合于自上而下 MS 分析的结构特异性肽。接下来，应用涉及shotgun 或靶向 MS 和无标记定量的蛋白质组学工作流程，直接在蛋白质组提取物中测量结构依赖性蛋白水解模式。LiP-MS 的可能应用包括发现扰动诱导的蛋白质结构改变、鉴定药物靶点、检测与疾病相关的蛋白质结构状态以及直接在生物样本中分析蛋白质聚集体。该方法还能够鉴定参与结构转变或受结合事件影响的特定蛋白质区域。样品制备大约需要 2 天，然后根据分析的样品数量，需要 1 到几天的 MS 和数据分析时间。具有基本生物化学培训背景的科学家可以实施样品制备步骤。MS 测量和数据分析需要蛋白质组学背景。
