Manfredi Marcello, Brandi Jessica, Conte Eleonora, Pidutti Paolo, Gosetti Fabio, Robotti Elisa, Marengo Emilio, Cecconi Daniela
Department of Sciences and Technological Innovation, University of Piemonte Orientale, Viale T. Michel 11, 15121, Alessandria, Italy; ISALIT S.r.l., Novara, Italy.
Department of Biotechnology, Proteomics and Mass Spectrometry Lab, University of Verona, Strada le Grazie 15, 37134, Verona, Italy.
Anal Biochem. 2017 Nov 15;537:72-77. doi: 10.1016/j.ab.2017.08.021. Epub 2017 Aug 30.
We conducted a proteomics study in order to detect the proteomic method which provides the most complete characterization of the proteins of rice milk. In particular, we compared the results obtained from LC-MS/MS after protein precipitation with acetone or TCA, as well as the results obtained from LC-MS/MS after protein prefractionation based on SDS-PAGE (GeLC-MS/MS) or ProteoMiner™ technology (ProteoMiner-LC-MS/MS), and after peptide prefractionation based on IEF (pIEF-LC-MS/MS). A total of 158 protein species have been detect in rice milk. The physical-chemical analysis and classification of the identified proteins were also reported. In particular, we showed that pIEF-LC-MS/MS method led to a significant increase in the proteome coverage, allowing the identification of a total of 96 proteins of milk rice. This study demonstrates the utility of a prefractionation step based on pIEF before the shotgun proteomic analysis and offers an in-depth insight into the rice milk proteome.
我们开展了一项蛋白质组学研究，以检测能对米乳蛋白质进行最全面表征的蛋白质组学方法。具体而言，我们比较了蛋白质经丙酮或三氯乙酸沉淀后进行液相色谱-串联质谱（LC-MS/MS）分析所得结果，以及基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）的蛋白质预分级（凝胶内酶解液相色谱-串联质谱，GeLC-MS/MS）或ProteoMiner™技术（ProteoMiner-LC-MS/MS）后进行LC-MS/MS分析所得结果，还有基于等电聚焦（pIEF-LC-MS/MS）的肽段预分级后进行LC-MS/MS分析所得结果。在米乳中共检测到158种蛋白质。还报告了所鉴定蛋白质的理化分析和分类情况。具体而言，我们发现pIEF-LC-MS/MS方法能显著提高蛋白质组覆盖率，共鉴定出96种米乳蛋白质。本研究证明了在鸟枪法蛋白质组分析之前基于pIEF进行预分级步骤的实用性，并深入洞察了米乳蛋白质组。
