McAtee C P, Zhang Y, Yarbough P O, Fuerst T R, Stone K L, Samander S, Williams K R
Genelabs Technologies, Inc., Redwood City, CA 94063, USA.
J Chromatogr B Biomed Appl. 1996 Oct 11;685(1):91-104. doi: 10.1016/0378-4347(96)00143-0.
A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.
一种分子量约为62.10(3)、源自戊型肝炎病毒（HEV：缅甸株）开放阅读框2（ORF-2）的蛋白质，在杆状病毒表达载体中表达并纯化至同质。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，重组62 kDa蛋白似乎是一个双峰。胰蛋白酶消化结合激光解吸质谱（LD-MS）以及胰蛋白酶肽段的序列分析表明，氨基末端被封闭，尽管未发生蛋白水解降解。所确定的肽段内部序列与预测的ORF-2蛋白一致。反相液相色谱与电喷雾质谱联用（LC-MS）将双峰蛋白解析为分子量分别为56548.5和58161.4的两个主要成分。通过LD-MS后离子衰变对分子氨基末端的确认，使我们能够初步将每个物种的羧基末端定位在第540和525位残基处。通过自动羧基末端测序对完整蛋白进行测序，证实羧基末端被截断，且LC-MS预测的序列定位是正确的。
