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Sequencing the prion protein using microwave assisted acid hydrolysis combined with MALDI-MS Reiz, Béla1, A. Lo1, D. Duggan2, B. Suriyamongkol2, D. Wishart2,3, L. Li1 1 Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9 3 Department of Computing Science, University of Alberta, Edmonton, Alberta T6G 2E8 Introduction a) b) c) d) The ability of mass spectrometry to precisely identify proteins, peptides and modified amino acids makes it a powerful and indispensable technique in protein-related research. Our project involves the use of mass spectrometry to identify solvent exposed residues. In order to probe the structure of the prion protein we use chemical modification methods followed by the hydrolysis or proteolysis of the protein to identify modified residues. The analysis is carried out by using liquid chromatography (LC) e) combined with matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). a Experimental We use an enzyme- and detergent-free approach for the Figure 2. a) MALDI spectrum of the wild type prion construct; b-e) MALDI spectra after mixing with 6M HCl and microwaving for b) 1 min, c) 2 min, d-e) 3 min hydrolysis of the wild-type prion construct. The experimental flow is presented in Figure1. The protein was denatured by heat at 95°C for 5 minutes. Future Work Dithiothreithol (DTT) was added to reduce cysteines. The presence of dithiothreithol prevents oxidation of methionine and Figure 1. Experimental workflow Optimization of the microwave assisted acid hydrolysis process. tryptophan. The digestion was performed by mixing 10 mL of protein solution Optimization of the protocol for different prion constructs. Results and Discussion Develop a MALDI-MS/MS protocol to determine the surface aqueous solutions. After 1 minute of irradiation time (b) the protein was mostly intact. exposure of different residues in the prion construct. The samples were irradiated for various times. After 2 minutes of irradiation time (c) more peptide peaks were detected but the The sample was dried and resuspended in 0.1% trifluoroacetic intensity of these peaks compared to the intensity of the protein peaks was low. Acknowledgement acid. After 3 minutes of irradiation time (d and e) many low mass peptides are This work was funded by Alberta Prion Research Institute and Samples were spotted using a 2-layer method on a MALDI plate generated with relative intensities higher than that of the intact protein. PrioNet Canada. with 10 mL of 6M HCl or 50 % trifluoroacetic acid (TFA) (v/v) and analyzed by MALDI-MS.