Sandoval Wendy N, Pham Victoria, Ingle Elizabeth S, Liu Peter S, Lill Jennie R
Protein Chemistry Department, Genentech Inc., South San Francisco, CA 94080, USA.
Comb Chem High Throughput Screen. 2007 Nov;10(9):751-65. doi: 10.2174/138620707783018504.
Biotechnology has recently celebrated 30 years both as a science and as a multi-billion dollar industry. One application of biotechnology is to use human genetic information to discover, develop, manufacture, and commercialize biotherapeutics. Recombinant proteins can be produced in large quantities at high purity. High-throughput proteomic analysis is at the heart of the biotechnology research and development process, and the industry is constantly striving to streamline and automate the analytical processes involved. Microwave-assisted proteomics has recently emerged as a tool for increasing the bio-catalysis of several processes including tryptic digestions lipase selectivities, identification of metal-catalyzed oxidation sites on proteins, identification of protein N- and C-termini and enzyme catalyzed N-linked deglycosylation. Here, we explore the above mentioned methods, and describe our experiences evaluating microwave-technology for other common proteomic protocols including: removal of N-terminal pyroglutamyl for antibody characterization, beta elimination and Michael addition for identification of phosphorylation sites on recombinant proteins and enzyme mediated O-linked deglycosylation.
生物技术作为一门科学和一个价值数十亿美元的产业,最近迎来了它的30周年。生物技术的一个应用是利用人类遗传信息来发现、开发、生产和商业化生物治疗药物。重组蛋白可以大量生产且纯度很高。高通量蛋白质组学分析是生物技术研发过程的核心,该行业也在不断努力简化和自动化所涉及的分析过程。微波辅助蛋白质组学最近已成为一种工具,可用于增强包括胰蛋白酶消化、脂肪酶选择性、蛋白质上金属催化氧化位点的鉴定、蛋白质N端和C端的鉴定以及酶催化的N-连接去糖基化等多个过程的生物催化作用。在此,我们探讨上述方法,并描述我们在评估微波技术用于其他常见蛋白质组学方案方面的经验,这些方案包括:去除N端焦谷氨酸用于抗体表征、β消除和迈克尔加成用于鉴定重组蛋白上的磷酸化位点以及酶介导的O-连接去糖基化。