Baghalabadi Venus, Doucette Alan A
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, B3H 4R2, Canada; Department of Chemistry, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, P.O. Box 53714-161, Tabriz, Iran.
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, B3H 4R2, Canada.
Anal Chim Acta. 2020 Nov 22;1138:38-48. doi: 10.1016/j.aca.2020.08.057. Epub 2020 Sep 2.
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 ZnSO 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.
基于溶剂的蛋白质沉淀具有出色的回收率，尤其是在溶液的离子强度得到控制时。虽然沉淀非常适合在质谱分析之前进行完整蛋白质的纯化，但低分子量（LMW）蛋白质和肽在有机溶剂中被认为不易聚集。由于盐和有机溶剂（如丙酮）的组合尚未用于沉淀LMW蛋白质，因此我们在此确定基于溶剂的蛋白质沉淀的低质量极限。我们建立了用于LMW蛋白质和肽高回收率沉淀的优化条件。我们的结果表明，从复杂混合物中回收LMW成分对盐的类型有很强的依赖性。加入100 mM硫酸锌和97%的丙酮可实现低至2 kDa的所有肽的近定量回收，对于分子量为1 kDa的肽，回收率继续超过90%。通过自下而上的质谱分析，对复杂系统中胰蛋白酶和胃蛋白酶消化产生的沉淀肽进行了详细表征。
