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.

中文翻译：

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使用有限的蛋白水解耦合质谱法在整个蛋白质组范围内测量蛋白质结构变化。

由外部干扰或内部提示引起的蛋白质结构变化可深刻影响蛋白质活性，从而调节细胞生理。许多生物物理方法可用于探测蛋白质的结构变化，但不适用于生物提取物中的整个蛋白质组。有限的蛋白水解偶联质谱法（LiP-MS）是最近开发的一种蛋白质组学方法，可以在蛋白质组范围内直接在其复杂的生物学环境中鉴定蛋白质结构变化。在感兴趣的扰动之后，蛋白质组提取物经过在天然条件下应用非特异性蛋白酶的双重蛋白酶消化步骤，然后在变性条件下用序列特异性蛋白酶胰蛋白酶完全消化。这种顺序处理可产生适用于自下而上的MS分析的结构特异性肽。接下来，将涉及shot弹枪或靶向MS和无标记定量的蛋白质组学工作流程直接用于在蛋白质组提取物中测量与结构相关的蛋白质水解模式。LiP-MS的可能应用包括发现扰动引起的蛋白质结构改变，确定药物靶标，检测与疾病相关的蛋白质结构状态以及直接在生物样品中分析蛋白质聚集体。该方法还使得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，然后需要一到几天的MS和数据分析时间，具体取决于所分析样品的数量。经过基本生物化学培训的科学家可以执行样品制备步骤。MS测量和数据分析需要蛋白质组学的背景知识。