Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry
Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry
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DOI：
10.1021/ac035107u
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发表时间：
2004-04-01
影响因子：
7.4
通讯作者：
Zare, RN
中科院分区：
文献类型：
--
作者：
Kato, M;Sakai-Kato, K;Zare, RN
在熔融石英毛细管（i.d. 75 μ m）。采用溶胶-凝胶法制备了胃蛋白酶包埋膜。优化溶胶-凝胶反应，使得含有胃蛋白酶的溶胶溶液在最初在毛细管入口处制造的光聚合溶胶-凝胶（PSG）整料上形成薄膜。胃蛋白酶被封装到凝胶基质中而不失去其活性。PSG整体柱的大表面积使得固定化的胃蛋白酶能够实现高催化周转率，并且PSG的多孔性质促进大分子蛋白质渗透到柱中。固定化的胃蛋白酶消化的肽和蛋白质，以及所得的肽片段的混合物，可以直接分离在毛细管的部分，其中没有PSG整料存在。对制备的胃蛋白酶包被柱的耐久性和重复性进行了测试，结果令人满意。使用由0.5 M甲酸组成的酸性溶液作为运行缓冲液，因为它抑制蛋白质或肽在毛细管内表面上的吸附，并且能够将毛细管电泳柱的输出直接连接到质谱仪。胰岛素链β和溶菌酶的在线消化提供了蛋白水解肽的鉴定。胰岛素链β氨基酸序列的回收率为100%，溶菌酶氨基酸序列的回收率为73%。
A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 mum) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.