Jmeian Yazen, El Rassi Ziad
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071, USA.
J Proteome Res. 2007 Mar;6(3):947-54. doi: 10.1021/pr060660o. Epub 2007 Feb 10.
Affinity monolithic microcolumns with immobilized affinity ligands including protein A, protein G' and polyclonal antibodies were developed for the microscale depletion of the top eight most abundant proteins in human serum. These various affinity microcolumns were evaluated for their sample loading capacities with the standard protein substrates. In general, the sample loading capacity of protein A and protein G' was about 7-25 fold higher than that of the antibody-based affinity columns. The macroporous nature of the monolithic columns, which offers high permeability in pressure-driven flow, allowed the design of long tandem affinity columns for the simultaneous depletion of the top eight most abundant proteins in a single run. The tandem format could be extended to include additional affinity monolithic columns to deplete other proteins for which specific antibodies are available without running into high inlet pressure. Furthermore, the tandem affinity columns were integrated with immobilized trypsin monolithic columns to achieve the simultaneous depletion and digestion of proteins. The various formats investigated in this study could be down scaled to achieve nanoLC or up scaled to perform conventional HPLC depending on the size of the proteomic samples.
开发了具有固定化亲和配体（包括蛋白A、蛋白G'和多克隆抗体）的亲和整体微柱，用于在微尺度上去除人血清中含量最高的前八种蛋白质。使用标准蛋白质底物评估了这些不同的亲和微柱的样品加载能力。一般来说，蛋白A和蛋白G'的样品加载能力比基于抗体的亲和柱高约7 - 25倍。整体柱的大孔性质在压力驱动流中具有高渗透性，这使得可以设计长串联亲和柱，以便在一次运行中同时去除含量最高的前八种蛋白质。串联形式可以扩展到包括额外的亲和整体柱，以去除有特异性抗体可用的其他蛋白质，而不会导致高入口压力。此外，串联亲和柱与固定化胰蛋白酶整体柱集成，以实现蛋白质的同时去除和消化。本研究中研究的各种形式可以根据蛋白质组学样品的大小进行缩小以实现纳升液相色谱（nanoLC），或者放大以进行传统的高效液相色谱（HPLC）。
