Protein Analytical Chemistry Department, Genentech, Incorporated, 1 DNA Way, South San Francisco, California 94080, USA.
Anal Chem. 2009 Dec 1;81(23):9744-54. doi: 10.1021/ac901498k.
Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals.
重组组织型纤溶酶原激活剂（rt-PA）是一种具有丰富文献记载的糖蛋白，其结构、翻译后修饰以及 O-和 N-糖基化已有大量报道。大部分的鉴定工作是在 20 世纪 80 年代末完成的。然而，在过去的 20 年里，质谱技术取得了突飞猛进的发展，在软电喷雾电离、速度、分辨率和质量测量精度方面提供了新的能力。从这个角度来看，使用现代分析技术的新功能重新研究熟悉的蛋白质（如 rt-PA）的特性是值得的。在这项工作中，我们应用带有最先进仪器的 LC-MS 对 rt-PA 的糖型进行了表征。该方法利用精确质量测量以及快速的“源内”电压切换，用于检测糖的特征氧鎓离子。该方法基于对 rt-PA 聚糖的现有知识，证实了先前鉴定的聚糖结构。此外，我们还在 rt-PA 中鉴定了两种新的聚糖结构。在非典型 Asn-Xaa-Cys 共识序列中检测到低水平的 Asn142 N-糖基化。发现它主要被双天线杂合结构修饰。使用最近开发的电子转移解离（ETD）技术证实了这个 N-糖基化位点。同样使用这种方法，我们检测到低水平的 fucose-O-Thr61 延伸到二、三、四糖，这在以前的 rt-PA 中没有观察到。结果表明，使用最先进的分析方法可以揭示先前未检测到的低水平修饰的熟知生物制药。
