Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Immobilised enzymes on customisable micro solid phase extraction cartridges for proteolytic digests in minutes (#260)

Karen Duong , Simin Maleknia , Andrew Minett , Philip Doble

The rapid identification and quantification of biomarkers are of increasing significance in clinical settings, and this is achievable on small sample sizes by integrating the ePrep customisable micro solid phase extraction devices (microSPEeD) with mass spectrometry. The ePrep microSPE cartridges are comprised of a packed sorbent bed compartment, a unidirectional valve and a simple press fit connector for use with a digital syringe drive or an automated sample preparation workstation. The sorbent materials of the microSPE cartridges (particles of 1.5-3µm) have been specifically functionalised (e.g. carboxyl substituted silica), which enable the cartridges to be customised as affinity separation cartridges or immobilised enzyme micro-reactors. The use of positive displacement digital syringes ensures precise and reproducible flow kinetics, providing the opportunity to perform time-dependent assays, such as speeding up protein digests to timescales of minutes.

This presentation will demonstrate the utility of cartridges as immobilized enzyme micro-reactors for protein digestion in minutes. Details for the immobilisation of trypsin as a proof-of-concept enzyme and protocols for the digestion of a series of standard proteins (e.g. bovine serum albumin (BSA, ~66 kDa) and lysozyme (~14 kDa)) will be illustrated. The immobilisation of trypsin on carboxyl substituted silica cartridge has been performed in minutes, which in turn enabled the rapid digestion of the proteins (up to ~50µg) in timescales of minutes. The protein digests were then analysed by liquid chromatography electrospray ionization time-of-flight mass spectrometry, providing automated mass mapping and confirming protein sequence coverage comparable to the conventional methods (i.e. overnight digests).