Fountoulakis M, Juranville J F, Röder D, Evers S, Berndt P, Langen H
Preclinical Central Nervous System Research Gene Technology, Switzerland.
Electrophoresis. 1998 Jul;19(10):1819-27. doi: 10.1002/elps.1150191046.
Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels does not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second-dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted laser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.
在传统的Tris-甘氨酸凝胶上通过二维聚丙烯酰胺凝胶电泳分析流感嗜血杆菌的蛋白质组，通常无法有效分离5-20 kDa范围内的蛋白质，这些蛋白质主要聚集在较低的酸性和碱性区域。为了改善低分子量蛋白质的分离效果，我们在二维分离中使用了两种尿素浓度的均一性Tricine凝胶。Tricine凝胶系统能够有效且可重复地分离质量在5至20 kDa之间的微生物蛋白质，然而，质量低于5 kDa的蛋白质无法可视化。通过基质辅助激光解吸/电离-质谱法鉴定了约80种在5-25 kDa区域迁移的蛋白质，其中40种是首次鉴定。低质量蛋白质的消化通常只产生很少的肽段，不足以通过质谱法进行可靠鉴定。因此，偶尔通过先用胰蛋白酶或内肽酶Lys-C作为第一种酶，再用羧肽酶P作为第二种酶进行顺序消化来实现鉴定。所述凝胶系统可能有助于从其他生物体中有效分离低分子量蛋白质以构建标准图谱。
