PNNL

Abstract

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 on-line pressurized digestion system coupled to an ion mobility time of flight mass spectrometer and/or an ion trap mass spectrometer. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to 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 on-line digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research; for example, quick screening of protein-based drugs.