BIA Separations is now part of Sartorius. Learn more

Miniaturisation of trypsin immobilised enzymatic reactor for protein digestion

Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.

Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.