John H, Forssmann W G
IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, D-30625 Hannover, Germany.
Rapid Commun Mass Spectrom. 2001;15(14):1222-8. doi: 10.1002/rcm.367.
Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.
内皮抑素是胶原蛋白 XVIII 的 C 末端片段，是一种很有前景的蛋白质药物，因其具有抗血管生成活性而正在被开发用于癌症治疗。尽管已经发现了几种 N 末端长度和翻译后修饰不同（18.5 - 22 kDa）的人内皮抑素内源性分子形式，但在临床试验中仅使用了一种 20 kDa 的重组形式。这种在毕赤酵母中重组表达的蛋白质含有四个形成两个二硫键（Cys1 - Cys4 和 Cys2 - Cys3）的半胱氨酸。相比之下，关于内源性物质的二硫键模式存在相互矛盾的数据。本报告介绍了从人血液滤过液中分离出的内源性循环内皮抑素和重组蛋白的二硫键分析。通过在离线纳米喷雾模式下进行的埃德曼降解、基质辅助激光解吸/电离飞行时间质谱（MALDI - TOFMS）和电喷雾电离离子阱质谱（ESI - ITMS）来确定二硫键模式。所有天然和重组内皮抑素均表现出 Cys1 - Cys4（Cys(162) - Cys(302)）和 Cys2 - Cys3（Cys(264) - Cys(294)）连接。为了清晰讨论从 MS(n) 实验中获得的片段化二硫键连接的肽链，提出了一种改进的通用命名法。
