(493f) Fast on–Line Digestion System for High-Throughput Proteomics | AIChE

(493f) Fast on–Line Digestion System for High-Throughput Proteomics


López-Ferrer, D. - Presenter, Pacific Northwest National Laboratory

Within the last decade, the field of proteomics has benefited biological research as well as, the pharmaceutical, food and biotech industries. The analysis pipeline is based in a bottom up approach, where proteins are digested into peptides using trypsin as enzyme, and the resulting peptides are then analyzed by liquid chromatography (LC)-mass spectrometry (MS). However, the enzymatic digestion is one of the most time-consuming steps in the analysis workflow as it is typically carried out manually. We present an efficient on-line digestion system that reduces the number of sample manipulation steps 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, an ultra-fast 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 a mass spectrometer. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to high-throughput applications. 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.