Prions are composed largely, if not entirely, of prion protein (PrPsc in the case of scrapie). Although the formation of PrPs from the cellular prion protein (PrPc) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPsc synthesis. To assess this possibility, we purified both PrPC and PrPsc by using nondenaturing procedures and determined the secondary structure ofeach. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high a-helix content (42%) and no (3sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the -sheet content of PrPSc was 43% and the a-helix

In vivo under pathological conditions, the normal cellular form of the prion protein, PrP C (residues 23-231), misfolds to the pathogenic isoform PrP Sc , a ␤-rich aggregated pathogenic multimer. Proteinase K digestion of PrP Sc leads to a proteolytically resistant core, PrP 27-30 (residues 90 -231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP Sc and found that it can adopt two non-native abnormal isoforms, a ␤-oligomer and an amyloid fibril. Several lines of kinetic data suggest that the ␤-oligomer is not on the pathway to amyloid formation. The preferences for forming either a ␤-oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the ␤-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high ␤-sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct ␤-sheet-rich abnormal isoforms may explain the difficulties in refolding PrP Sc in vitro, the need for a PrP Sc template, and the significant variation in disease presentation and neuropathology.

A novel ionization source for biological mass spectrometry is described that combines atmospheric pressure (AP) ionization and matrix-assisted laser desorption/ionization (MALDI). The transfer of the ions from the atmospheric pressure ionization region to the high vacuum is pneumatically assisted (PA) by a stream of nitrogen, hence the acronym PA-AP MALDI. PA-AP MALDI is readily interchangeable with electrospray ionization on an orthogonal acceleration time-of-flight (oaTOF) mass spectrometer. Sample preparation is identical to that for conventional vacuum MALDI and uses the same matrix compounds, such as alpha-cyano-4-hydroxycinnamic acid. The performance of this ion source on the oaTOF mass spectrometer is compared with that of conventional vacuum MALDI-TOF for the analysis of peptides. PA-AP MALDI can detect low femtomole amounts of peptides in mixtures with good signal-to-noise ratio and with less discrimination for the detection of individual peptides in a protein digest. Peptide ions produced by this method generally exhibit no metastable fragmentation, whereas an oligosaccharide ionized by PA-AP MALDI shows several structurally diagnostic fragment ions. Total sample consumption is higher for PA-AP MALDI than for vacuum MALDI, as the transfer of ions into the vacuum system is relatively inefficient. This ionization method is able to produce protonated molecular ions for small proteins such as insulin, but these tend to form clusters with the matrix material. Limitations of the oaTOF mass spectrometer for singly charged high-mass ions make it difficult to evaluate the ionization of larger proteins.

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.

The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification; however, we cannot eliminate the possibility that a small fraction of PrPSc is modified by an as yet unidentified posttranslational process or that PrPC carries a modification that is removed in the formation of PrPSc. It seems likely that PrPSc differs from PrPC in its secondary and tertiary structure, but the possibility of a tightly bound, disease-specific molecule which purifies with PrPSc must also be considered.