On-line Digestion System for Protein Characterization and Proteome Analysis

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• 作者：Daniel Lpez-Ferrer ; Konstantinos Petritis ; Natacha M. Lourette ; Brian Clowers ; Kim K. Hixson ; Tyler Heibeck ; David C. Prior ; Ljiljana Paa-Toli ; David G. Camp ; ; Mikhail E. Belov ; Richard D. Smith
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：December 1, 2008
• 年：2008
• 卷：80
• 期：23
• 页码：8930-8936
• 全文大小：377K
• 年卷期：v.80,no.23(December 1, 2008)
• ISSN：1520-6882

文摘

An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research (rapid screening of protein-based drugs).