(416d) Accelerating Enzymatic Reactions in Proteomics Using Unconventional Energy Inputs

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 on a bottom up approach, where proteins are digested into peptides using the protease trypsin, and the resulting peptides are then analyzed by liquid chromatography (LC)-mass spectrometry (MS). Of all processes in the workflow, the enzymatic digestion is one of the most time-consuming and labor intensive steps. We have recently found that applying high pressures to trypsin digestions is very effective in hastening the proteolytic activity of trypsin, which translates into reduced sample preparation times. We have studied the role of elevated pressures on enzymatic digestions for proteomic applications both on-line and off-line with detection of the resultant peptides obtained by LC-MS. For the off-line application, we used a Barocycler?, which applies pressure cycling technology (PCT) in the range of 0 to 35 kpsi to a sample. The PCT was shown to be able to rapidly digest proteins as well as label peptides with 18O. The performance was evaluated in terms of the labelling efficiency of the peptides. In order to improve the enzyme kinetics further and to move towards robust and long lasting enzyme recycling, the concomitant use of pressure and immobilized trypsin-coated magnetic nanoparticles was explored. In addition, in order to explore the possibility of an automatable sample preparation system, an on-line digestion system was developed which allows for cell lysis, protein derivatization, protein digestion and mass spectrometric analysis all in one analysis run. The system reduces the number of sample manipulation steps making it ideal for increased reproducibility and quantification of low-abundance proteins. To push experimental possibilities further we explored the integration of glycomics and proteomics studies within the same system modifying our on-line trypsin digestion system to incorporate deglycosylation reactions as well. This multiplexed enzyme reactor/digester, which uses pressurized solvents to enhance desired enzyme kinetics looks promising for ultra-high-throughput applications, i.e., ?omics on the fly.?