Solvent-based protein precipitation provides exceptional recovery, particularly when the ionic strength of the solution is controlled. While precipitation is ideally suited for intact protein purification ahead of mass-spectrometry, low molecular weight (LMW) proteins and peptides are considered less susceptible to aggregation in organic solvent. As the combination of salt and organic solvent (i.e. acetone) has yet to be exploited to precipitate LMW proteins, we herein determine the low mass limit for solvent-based protein precipitation. We establish optimized conditions for high recovery precipitation of LMW proteins and peptides. Our results demonstrate a strong dependence on the type of salt to recover LMW components from complex mixtures. Inclusion of 100 mM ZnSO4 with 97% acetone provides near quantitative recovery of all peptides down to 2 kDa, and continues to exceed 90% yield for peptides at a molecular weight of 1 kDa. A detailed characterization of the precipitated peptides resulting from trypsin and pepsin digestion of complex systems is provided by bottom-up mass spectrometry.

中文翻译：

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通过丙酮沉淀分离的低分子量蛋白质和肽的质谱分析

溶剂型蛋白质沉淀可提供出色的回收率，尤其是在控制溶液的离子强度时。虽然沉淀非常适合在质谱分析之前纯化完整的蛋白质，但低分子量 (LMW) 蛋白质和肽被认为不太容易在有机溶剂中聚集。由于尚未利用盐和有机溶剂（即丙酮）的组合来沉淀 LMW 蛋白质，我们在此确定基于溶剂的蛋白质沉淀的低质量限制。我们为 LMW 蛋白质和肽的高回收率沉淀建立了优化条件。我们的结果表明，从复杂的混合物中回收 LMW 组分强烈依赖于盐的类型。包含 100 mM ZnSO4 和 97% 丙酮，可对低至 2 kDa 的所有肽提供接近定量的回收率，并且对分子量为 1 kDa 的肽的收率继续超过 90%。自下而上质谱法提供了复杂系统的胰蛋白酶和胃蛋白酶消化产生的沉淀肽的详细表征。