Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

中文翻译：

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通过质谱法进行蛋白质组分析的快速和定量蛋白质沉淀。

蛋白质沉淀是蛋白质组分析以及其他应用（例如，用于小分子分析的蛋白质消耗、蛋白质的批量商业制备）的常见前端制备策略。用于沉淀蛋白质的高度可变的条件，包括溶剂类型、强度、时间和温度，反映了不一致和低回收率。因此，不完整的蛋白质组覆盖会降低沉淀在质谱分析之前用于蛋白质组样品制备的效用。我们在此研究和优化通过使用丙酮在确定的离子强度下沉淀影响蛋白质回收的条件。通过使用 80% 丙酮提高盐浓度和孵育温度，我们表明，快速（2 分钟）沉淀可提供始终如一的高蛋白质回收率（98 ± 1%）的复杂蛋白质组提取物。快速沉淀也适用于分离低至 1 μg mL-1 的稀释蛋白质。此外，通过自下而上质谱 (MS) 对蛋白质颗粒的分析揭示了所有蛋白质在分子量、等电点 (pI) 和疏水性方面的无偏回收率。我们强大的蛋白质分离策略最大限度地提高了回收率和通量，利用了沉淀相对于替代技术的分析优势。数据可通过 ProteomeXchange 获得，标识符为 PXD015674。通过自下而上的质谱 (MS) 对蛋白质颗粒的分析揭示了所有蛋白质在分子量、等电点 (pI) 和疏水性方面的无偏回收率。我们强大的蛋白质分离策略最大限度地提高了回收率和通量，利用了沉淀相对于替代技术的分析优势。数据可通过 ProteomeXchange 获得，标识符为 PXD015674。通过自下而上的质谱 (MS) 对蛋白质颗粒的分析揭示了所有蛋白质在分子量、等电点 (pI) 和疏水性方面的无偏回收率。我们强大的蛋白质分离策略最大限度地提高了回收率和通量，利用了沉淀相对于替代技术的分析优势。数据可通过 ProteomeXchange 获得，标识符为 PXD015674。