Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Analyst. 2017 Jul 10;142(14):2578-2586. doi: 10.1039/c7an00778g.
Proteolysis is often a critical step in protein characterization via mass spectrometry. Compared to complete digestion, limited proteolysis gives larger peptides, and the dominant cleavage sites may identify highly accessible, flexible protein regions. This paper explores controlled proteolysis in porous nylon membranes containing immobilized trypsin. Passage of protein solutions through ∼100 μm thick membranes provides reaction residence times as short as milliseconds to limit digestion. Additionally, variation of the membrane pore size and the protease-immobilization method (electrostatic adsorption or covalent anchoring to adsorbed polymer in membrane pores) affords control over the proteolysis rate. When digesting the highly labile protein β-casein, large membrane pores (5.0 μm) and covalent enzyme anchoring to adsorbed polymer lead to particularly long tryptic peptides. With the more trypsin-resistant proteins cytochrome c and apomyoglobin, in-membrane proteolysis with short residence times, 1.2 μm membrane pores, and trypsin electrostatically immobilized to an adsorbed polyanion cleaves the proteins after lysine residues in flexible regions. For both cytochrome c and apomyoglobin, cleavages in an interhelix region yield two particularly large peptides that cover the entire protein sequence.
蛋白水解通常是通过质谱进行蛋白质特征分析的关键步骤。与完全消化相比，有限的蛋白水解可产生更大的肽，并且主要的切割位点可鉴定出高度可及的、灵活的蛋白质区域。本文探讨了在含有固定化胰蛋白酶的多孔尼龙膜中进行的可控蛋白水解。蛋白质溶液通过约 100 μm 厚的膜，反应停留时间短至毫秒，可限制消化。此外，改变膜的孔径和蛋白酶固定化方法（静电吸附或共价锚定到膜孔中吸附的聚合物）可以控制蛋白水解速率。在消化高度不稳定的蛋白质 β-酪蛋白时，大的膜孔（5.0 μm）和固定在吸附聚合物上的共价酶锚定导致特别长的胰蛋白酶肽。对于更耐胰蛋白酶的蛋白质细胞色素 c 和脱辅基肌红蛋白，在膜内进行短停留时间的蛋白水解、1.2 μm 的膜孔以及静电固定在吸附多阴离子上的胰蛋白酶，在柔性区域的赖氨酸残基后切割蛋白质。对于细胞色素 c 和脱辅基肌红蛋白，在螺旋间区域的切割产生了两个特别大的肽，覆盖了整个蛋白质序列。
