Location: Produce Safety and Microbiology Research
Title: Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure Author
Submitted to: Prion
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 16, 2014
Publication Date: February 7, 2014
Repository URL: http://www.tandfonline.com/doi/full/10.4161/pri.27891
Citation: Silva, C.J. 2014. Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure. Prion. 8(1):42-50. DOI: http://dx.doi.org/10.4161/pri.27891. Interpretive Summary: Prions are proteins that can convert a normal cellular protein into the prion shape. These new prions can convert more normal proteins into the prion shape, which eventually results in disease. This is why prions are called pathological proteins. Mass spectrometry was used to show that a prion and the normal cellular protein are identical molecules. They have identical chemical features and only differ in terms of their unique shapes. These differences in shape mean that some regions of the prion molecule are unable to react with chemicals while the same portions of the molecule in the normal cellular protein shape do react with those chemicals. These differences can be detected by mass spectrometry. They can also be detected by using antibodies. In this way the tools of chemistry can be used to detect and study the structure of prions.
Technical Abstract: Prions are molecular pathogens, able to convert a normal cellular prion protein PrPC into a prion PrPSc. The information necessary for this conversion is contained in the conformation of PrPSc. Mass spectrometry and small-molecule covalent reactions have recently been used to study prions. This work describes how these tools have been successfully applied to the study of prions. Mass spectrometry (MS) has played an important role in our understanding of the structural differences between PrPC and PrPSc. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10-18mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrPSc and GPI-anchorless PrPSc. It has also been used to study the quaternary structure of the PrPSc multimer. Small molecule reagents react differently with the same lysine in the PrPC conformation than in the PrPSc conformation. Such differences can be detected by Western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrPSc in the presence of PrPC without the need for proteinase K pretreatment. Small molecule reagents, in combination with Western blot detection, have been used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions.