Vitorino Rui, Krenkova Jana, Foret Frantisek, Domingues Pedro, Amado Francisco
Department of Chemistry, Mass Spectrometry Center, QOPNA, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal.
Methods Mol Biol. 2011;790:31-46. doi: 10.1007/978-1-61779-319-6_3.
Body fluids and body tissues have a myriad of peptides and proteins that, very often, the traditional methodologies of proteomics, such as conventional gel electrophoresis or mass spectrometry, are unable to characterize. We describe two protocols to characterize high molecular weight peptides (>3 kDa) and intact proteins involving on-line trypsin digestion, separation of the digests by nano-HPLC, and analysis by mass spectrometry using two different ionization sources (matrix-assisted laser desorption and electrospray ionization). These protocols have the advantage of promoting protein denaturation in an aqueous-organic solvent, which reduces the derivatization of the sample and facilitates an in-depth analysis for detection and identification of proteins. Additional advantages include the following: (1) integration of these protocols into standard proteomic workflows after the preprocessing of samples and separation; (2) use of high-resolution monolithic columns; and (3) the ability to acquire information from minimal amounts of sample.
体液和身体组织含有无数的肽和蛋白质，而传统的蛋白质组学方法，如常规凝胶电泳或质谱分析，往往无法对其进行表征。我们描述了两种用于表征高分子量肽（>3 kDa）和完整蛋白质的方法，包括在线胰蛋白酶消化、通过纳米高效液相色谱分离消化产物，以及使用两种不同电离源（基质辅助激光解吸和电喷雾电离）进行质谱分析。这些方法的优点是能在水-有机溶剂中促进蛋白质变性，减少样品衍生化，并便于对蛋白质进行深入分析以进行检测和鉴定。其他优点包括：（1）在样品预处理和分离后，将这些方法整合到标准蛋白质组学工作流程中；（2）使用高分辨率整体柱；（3）能够从极少量样品中获取信息。
