Transcript Sequencing the prion protein using microwave assisted acid

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.