High-throughput mass spec protease assay to investigate the functional role of this enzyme class in the normal cellular function

Aim The aim of the project is to establish a 21st century approaches to the characterisation of protease activities in cellular extracts or body fluids. Proteolytic cleavage is now accepted as a key event in signal transduction (e.g., apoptotic cascade, NF-?B signalling) and in the governance of cellular metabolism. Furthermore the need for surrogate markers in clinical trials has heightened interest in protease, protein and peptide content of biological fluids. There is therefore increased interest in characterising protease activities in cellular function and extracellular fluids. However the available protease assays are still have a very low throughput. In this respect the low cost of drug assays in clinical laboratories, using multiple reaction monitoring (MRM) mass spectrometry (MS) may be a model that can be followed in protease assays. It is therefore our goal to develop a high-throughput protease assay at reasonable costs. Introduction Despite recent advances in the field, the substrates and in vivo roles for newly identified proteases are unknown and, even for proteases that have been well characterised, their biological functions are often not fully understood. New techniques are urgently required to identify the protease repertoire that is expressed and active in a cell, tissue or organism, as well as to identify as many natural substrates of each protease as possible by identifying substrate specificity in the primary sequence and then applying bioinformatics analysis to seek potential substrates. In the field of analytical chemistry, many small molecules (e.g., drug metabolites, hormones, protein degradation products) are routinely measured using this approach at high throughput with great precision (Coefficient of Variation or CV <5%). Most such assays employ electrospray ionisation followed by two stages of mass selection: a first stage (MS1) selecting the mass of the intact analyte (parent ion) and, after collision activated decomposition of the parent ion, a second stage (MS2) selecting a specific fragment of the parent, collectively generating a 'Selected Reaction Monitoring' (SRM) assay. In the Whetton lab a protocol for about 40 such transitions to be assessed on 40 different peptides has been used with great success in identifying specific post-translational modifications (Unwin et al 2005 Molecular and Cellular Proteomics 4: 1134-1144). This can be adapted to the project below. Workplan Year 0: On the award of the studentship a useful combination of synthetic peptide substrates, which provides potential substrates for all major classes of mammalian proteases will be synthesised. Year 1: Development of a sample assay workflow. Key elements include: definition of the components of the protease/substrate incubation suitable for downstream mass spectrometric assay. This will be followed by followed by peptide cleavage and product purification. Samples will be run on an AB QSTAR tandem mass spectrometer to define products. Information acquired on tandem MS profiles helps define specific fragment for MS2 in MRM assay. Years 2 & 3: Optimisation of the protease cleavage product purification procedure for a high-throughput approach. Determination of best MRM approach for each protease of interest using highly enriched proteases and defined peptide substrate library Translation of a selected sample assay workflow to an MRM based assay. Several hundred possible peptide products from protease reactions will be assessed with multiple MRM runs. Year 4: Testing of the developed high-throughput MRM assay for protease activity on mammalian cell culture extracts, cell culture supernatants as well as human body fluids like serum of healthy volunteers to validate the MRM protease multiplex assay and to characterize the protease activity content of these samples. As a standard reference to test for protease activity we will use array-based protease test systems.