Gorman J J, Shiell B J
Commonwealth Scientific and Industrial Research Organization, Australian Animal Health Laboratory, Geelong, Vic.
J Chromatogr. 1993 Aug 27;646(1):193-205. doi: 10.1016/s0021-9673(99)87021-6.
The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by "chymotryptic-like" cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.
强阳离子交换剂聚磺乙基天冬酰胺已被评估为一种从少量病毒蛋白的胰蛋白酶消化物中分离羧基末端和封闭氨基末端肽段的介质。带单个正电荷的肽段，如卵清蛋白和新城疫病毒（NDV）基质蛋白的封闭氨基末端肽段，以及卵清蛋白和NDV核衣壳蛋白的羧基末端肽段，在早期离子交换级分中洗脱，并通过随后的反相高效液相色谱很容易以均一形式分离出来。一些早期离子交换级分还包含通过“类胰凝乳蛋白酶样”裂解产生的单电荷肽段，而其他肽段因其高度酸性特征在这些级分中洗脱。带有额外碱性残基的末端序列从较晚洗脱的离子交换级分中分离出来。具有这种性质的肽段包括NDV核衣壳蛋白的封闭氨基末端和NDV基质蛋白羧基末端的一部分。本研究揭示了卵清蛋白氨基末端区域迄今未描述的多态性。还检测到了NDV基质蛋白羧基末端的截短肽段。这些肽段的存在可能反映了基质蛋白的羧基末端加工过程。本文所述策略对于从蛋白质的胰蛋白酶消化物中选择性微分离羧基末端肽段和封闭氨基末端肽段应具有普遍适用性。
