Pellets were resuspended in MiliQ H2O and Tricine buffer in a ratio 1:2. was then subjected to HPLC and the effluent fed to an ESI-TOF detector. The inset shows the total ion current LY2801653 dihydrochloride output. The MS spectrum of the peak in at 18.9C19.6 minutes is shown. Deconvolution of the spectrum yielded a main component with an average mass of 9400.76 Da (MH+), corresponding to a peptide with the Mo PrP sequence M153-S230 (theoretical average mass: 9399.5 Da). AN Additional species with an CTSD MH+ value of +17 LY2801653 dihydrochloride and likely corresponds to the same peptide with one of the three Met residues present in the sequence oxidized. A minor component with an MH+ value of 9531.61 Da might correspond to N152-S230 (theoretical average mass: 9513.6) with an oxidized methionine a component with M = 9514 Da.(TIF) ppat.1006797.s002.tif (1.6M) GUID:?83D73CD3-D4B3-418B-8B99-5F338A213D17 S3 Fig: Sequences of mouse and bank vole PrP. Secondary structure of PrPC, as determined by NMR studies (Mouse PDB 2L39; bank vole PDB 2K56): Lines placed on the PrPC sequence indicate the location of -sheet and Chelical regions.(TIF) ppat.1006797.s003.tif (3.2M) GUID:?098C0B08-CDEF-4ED6-936A-F7F9815B8D2F S4 Fig: Additional epitope mapping of PK-resistant peptides from recMoPrPSc-950. RecMoPrPSc-950 was treated with 10 g/ml PK as described in the Materials and methods sections, and under the same conditions used for the experiments described in Fig 3. Digested samples were subjected to Tris/Tricine SDS-PAGE, electroblotted and probed with monoclonal antibody SAF84, which recognizes epitope 166C172. SAF84, as expected, recognized the ~16 and ~14.6 kDa bands corresponding to 86/98-230 and 117/119-230 fragments, and more weakly, ~13, ~12, and ~10.2 kDa bands putatively corresponding to ~117/119-230, ~135C230, and ~152/153-230. Essentially no bands were detected with smaller apparent MW values, since they do not contain the (166C172) epitope. Also, none of the doubly truncated fragments detected by 3F10, which do not include the epitope, were detected.(TIF) ppat.1006797.s004.tif (792K) GUID:?59874811-46AB-495F-850E-96115E7044B7 S5 Fig: Histopathological and immunohistochemical analysis of brains from bank voles inoculated with recBVPrPSc. Two brain areas were investigated: striatium and retrosplenial cortex. Spongiform lesion was studied by haematoxylin-eosin (H&E) staining. PrPSc aggregates/deposits were observed by IHC staining (antibody 6C2). Finally astroglia activation was determined by GFAP staining.(TIF) ppat.1006797.s005.tif (7.2M) GUID:?AC7DBB81-5985-4F02-B26D-42DA60E52382 S6 Fig: MALDI spectra of tryptic digests of bands 1 to 5 of the gel shown in Fig 5, corresponding to PK-treated recBVPrPSc. The bands were excised and reduced, alkylated and digested in-gel with trypsin. Peptides were extracted, dried for 1 hat 4C (Sorvall ST 16R, Thermo Scientific). The resulting pellet was resuspended in 10 l PBS (Fisher Bioreagents) and stored at -20 C until microscopic examination. The sample was applied directly to a carbon-coated grid, R 2/2 Quantifoil? (Quantifoil), and rapidly plunged into liquid ethane using a Vitrobot (Maastricht Instruments BV). Sample analysis was performed with a JEM 2200F (JEOL) transmission cryo-electron microscope, using an acceleration voltage of 200 KV and defocus ranging from 21.2 to 22.5 mm, decided accurately by using enhanced power spectra. Images were obtained with a 2k62k TM Ultrascan 1000 CCD camera (Gatan).Images show short, untwisted fibrils, ~10 nm wide, apparently composed of two intertwined protofilaments, very similar to those of GPI-anchorless PrPSc [26]. Rosettes are glycogen present as a contaminant in the liver RNA extract used as part of the conversion mixture, as described before (Timmes et al. PLoS One. 2013 Jul 30;8(7):e71081. doi: 10.1371/journal.pone.0071081). (TIF) ppat.1006797.s007.tif (8.1M) GUID:?472CEB05-9695-4F1D-807A-1D77342B997A Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Very solid evidence suggests that the core of full length PrPSc is usually a 4-rung -solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the -strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133C134, 141, 152C153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of -strands, helping us to predict the threading of the -solenoid. We have now extended LY2801653 dihydrochloride this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of.