Author
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Onisko, Bruce |
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Submitted to: Mass Spectrometry
Publication Type: Abstract Only Publication Acceptance Date: 3/19/2003 Publication Date: 1/20/2003 Citation: Onisko, B.C. 2002 Analysis of recombinant hamster prion protein by "mass spectrometry".. Mass Spectrometry. Interpretive Summary: Technical Abstract: It is the purpose of the study to obtain lysine-lysine distance constraints by use of chemical cross-linking, endoproteolytic cleavage, and analysis of reaction products by tandem mass spectrometry on a Q-TOF geometry instrument obtained will be used to evaluated the validity of the current three dimensional model of PrPC based on NMR data. Recombinant hamster PrP protein will be cross linked with the lysine specific reagent, bis(sulfosuccinimidyl) suberate which has previously been shown to cross link lysines in proteins only if the through-space interatomic distance between the two alpha carbons of the lysines of interest are within 24 angstroms of each other. Following the cross linking reaction, the extent of derivatization will be monitored by electrospray mass spectrometry. Next, cysteine disulfides will be reduced with dithiothreitol and alkylated with iodoacetamide. Following disulfide reduction and alkylation, the derivatized protein will be proteolytically digested with trypsin, which specifically cleaves at the C-terminus of arginine and any remaining un-derivatized lysine residues. The proteolytically digested protein will be then analyzed by ESI-TOF and MALDI-TOF to determine the molecular weights of product peptides to mass accuracy of better than 30ppm. These molecular weights will then be compared to all possible cross-linked peptides. (Hamster PrP has (11) eleven lysines and (11) eleven arginines, so there are potentially 22X22 = 484 cross-linked tryptic peptides). Tentative assignments of cross linked peptides will be confirmed by obtaining and interpreting tandem mass spectrometric data from both ESI-Q-TOF and MALDI-Q-TOF experiments. (The working range for MALDI-Q-TOF is about molecular weight 2,500 because of the m/z limitation of the quadruple mass analyzer and the fact that MALDI products primarily singly charged ions. Products above MW 2,500 can be analyzed by electrospray since this ionization method multiplies charges proteins and peptides). Interatomic distance between all lysines in the three dimensional structure of hamster PrP will be calculated using Protein Explorer, and a list of all possible pairs of lysines that meet the distance constrain for bis (sulfosuccinimidyl) suberate will be tabulated and compared to the experimental set of cross linked peptides observed by mass spectrometry. |
