Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, via Belmeloro 6, 40126 Bologna, Italy; Center for Applied Biomedical Research (C.R.B.A.), S. Orsola-Malpighi Hospital, via Massarenti 9, 40138 Bologna, Italy.
Bia Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
Talanta. 2017 May 15;167:143-157. doi: 10.1016/j.talanta.2017.02.016. Epub 2017 Feb 5.
Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5mm×5.2mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90±9s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.
在开发基于质谱（MS）的蛋白质组学可靠分析平台时，减少实验变异性、限制污染和提高自动化水平是至关重要的目标。在这项工作中，通过共价固定在对流相互作用介质（CIMac™）分析柱（5mm×5.2mm ID）上，开发了新型基于胰蛋白酶的整体固定化酶反应器（trypsin-based monolithic immobilized enzyme reactors，tryp-IMERs）。尽管尺寸较小，但柱格式允许插入普通高效液相色谱（HPLC）系统，从而避免使用昂贵的微或纳米平台。优化了整体的孔径和表面化学性质，以实现即使在高分子量蛋白质存在的情况下也能达到高效的消化效率，并且避免蛋白质/肽的吸附、峰展宽和样品损失。对 tryp-IMERs 进行了全面的表征，以选择最佳的制备方案和胰蛋白酶类型。通过离线方法优化操作和储存条件。通过设置多维分析平台进行在线研究，该平台包括 tryp-IMER、捕集柱、分析 C4 柱和高分辨率混合质谱仪（ESI-Q-TOF）。在优化条件下，对于五种不同来源的不同分子量、等电点和可及性的切割位点的代表性样品蛋白（细胞色素 c、肌红蛋白和白蛋白），以及 200ng 的蛋白混合物，可实现快速的蛋白消化（90±9s）、高蛋白覆盖率（≥60%）和高得分值。表现最佳的 tryp-IMERs 显示出低至皮摩尔级别的高灵敏度。该平台还适用于分析高分子量蛋白，如人免疫球蛋白 G（hIgG）的混合物，以及人血浆蛋白的高分子量部分，其在不到两分钟的时间内被消化，程度与经典溶液内孵育方案中过夜孵育相当。最后，在研究过程中还突出了一些被低估的关键程序问题。这些方面对于 tryp-IMER 用户和 tryp-IMER 开发人员都具有普遍的兴趣。
