Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Anal Chem. 2012 Oct 2;84(19):8357-63. doi: 10.1021/ac3019153. Epub 2012 Sep 21.
Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 μm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.
在多孔尼龙中，聚（苯乙烯磺酸盐）（PSS）和蛋白酶的顺序吸附可产生用于有限蛋白质消化的酶膜反应器。尽管膜中的局部酶密度高（约 30mg/cm³）且孔径小导致在<1s 内进行消化，但膜的薄度（170μm）可以控制毫秒级的停留时间，以限制消化。脱铁细胞血红蛋白的消化表明，肽的长度随着在膜中的停留时间的减少而增加。此外，对大的肌红蛋白蛋白酶解肽（8kDa）进行电子转移解离（ETD）串联质谱（MS/MS）分析可提供 1-2 个氨基酸的分辨率。在变性条件下，牛血清白蛋白（BSA）的有限膜消化以及随后的 ESI-Orbitrap MS 分析显示出大的肽（3-10kDa），从而将序列覆盖率从 53%（2s 消化）提高到 82%（0.05s 消化）。通过这种方法，我们还对大型拟南芥 GTPase，根毛缺陷 3（RHD3）进行了基于膜的有限蛋白水解，并对靠近 C 末端的不稳定区域进行了适当的探测，这表明蛋白质重建可能使 RHD3 更适合结晶。
