Ecular Weight (Sigma-Aldrich, St. Louis, MO, USA), Kaleidoscope Prestained Standard (BioRad, Hercules, CA, USA) and Novex Sharp Protein Standard (Invitrogen, Carlsbad, CA, USA) were run in each analysis to calibrate the MW of the bands.with proteinase K, washed again, and then incubated overnight with the antibody 6H4 (1:2000, Prionics AG, Schlieren, Switzerland). The sections were developed using the DAKO EnVision system and 3,39diaminobenzidine as the chromogenic substrate.Supporting InformationFigure S1 Western blot of unpurified GPI2 PrPSc 2/+Mass SpectrometryNanoLC/ESI/MS analysis was done with an 15900046 Applied Biosystems (AB SCIEX, Framingham, MA) model QStar Pulsar equipped with a Proxeon Biosystems (Odense, Madrasin Denmark) nanoelectrospray source. Samples of the Gnd stock solution (vide supra) were loaded automatically onto a C-18 trapping cartridge and chromatographed on a reversed-phase column (Vydac Everest 238EV5.07515, 75 mm 6 150 mm) fitted with a coated spray tip (FS360-50-5-CE; New K162 chemical information Objective, Inc.). A nanoflow LC system (Dionex, Sunnyvale, CA) with autosampler, column switching device, loading pump, and nanoflow solvent delivery system was used. Elution solvents were A (0.5 acetic acid in water) and B (0.5 acetic acid in 80 acetonitrile/20 water). Samples were eluted at 250 nL/min using a binary gradient (8 B at 0 min to 80 B in a 30 min linear gradient, held at 80 B for 5 min, then back to 8 B for 15 minutes). The QStar Pulsar was externally calibrated daily with human [Glu1]-fibrinopeptide B. In parallel, 1 mL of the Gnd stock solution was mixed with with 49 mL of sinapinic acid (SA) solution (10 mg/mL SA dissolved in 30 ACN with 0.3 TFA) and analyzed by MALDI-TOF. One half mL aliquots were deposited using the dried-droplet method onto a 384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA). MALDI analysis was performed in a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems, Foster City, CA, USA). MS spectra were acquired in linear mode (20 kV source) with a Nd:YAG, (355 nm) laser, and averaging 500 laser shots. The mass of the peptide M153-S232 (9573 Da) was determined by an iterative calibration 
approach, using insulin (m/z = 5733), ribonuclease A (m/z = 13682) and lysozyme (m/ z = 14305), (Sigma-Aldrich, St. Louis, MO) as internal standards. Then, the signals from the M153-S232 (9573 Da), G89-S232 (16371 Da), and G81-S232 (17148 Da) peptides were used to calibrate the rest of peaks in the spectrum. Masses were matched to PrP fragments with the help of GPMAW 6.0 1326631 software (Lighthouse, Odense, Denmark).PK. Both samples were treated with PNGase F. WB was probed with the #51 antibody. (TIF)Figure S2 Characterization of isolated GPI2 PrPSc. 10 ml of sample were loaded and separated in a 15 gel by SDS-PAGE. The gel was stained by Coomassie blue. The molecular weight of the GPI-less PrP27-30 is ,16750 Da. (TIF) Figure S3 Western blot of PK-resistant fragments. In unpurified (1) and purified GPI- PrPSc (2). Both samples were digested with proteinase K, 25 mg/ml and 10 mg/ml, respectively, treated with PNGase F and resolved on a Tricine-SDS-PAGE gel. WB was probed with the R1 antibody. (TIF) Figure S4 Bayesian protein reconstruction of the nanoLC-ESI-MS spectra of PK-treated purified GPI2 PrPSc. The mass graphs of the three peaks: 17148 Da (top), 16729 Da (middle) and 16371 Da (bottom), identified by ESI-TOF are shown. (TIF) Figure S5 Western blot of recombinant MoPrP(23?31)cleavage by PK. Samples were digested with.Ecular Weight (Sigma-Aldrich, St. Louis, MO, USA), Kaleidoscope Prestained Standard (BioRad, Hercules, CA, USA) and Novex Sharp Protein Standard (Invitrogen, Carlsbad, CA, USA) were run in each analysis to calibrate the MW of the bands.with proteinase K, washed again, and then incubated overnight with the antibody 6H4 (1:2000, Prionics AG, Schlieren, Switzerland). The sections were developed using the DAKO EnVision system and 3,39diaminobenzidine as the chromogenic substrate.Supporting InformationFigure S1 Western blot of unpurified GPI2 PrPSc 2/+Mass SpectrometryNanoLC/ESI/MS analysis was done with an 15900046 Applied Biosystems (AB SCIEX, Framingham, MA) model QStar Pulsar equipped with a Proxeon Biosystems (Odense, Denmark) nanoelectrospray source. Samples of the Gnd stock solution (vide supra) were loaded automatically onto a C-18 trapping cartridge and chromatographed on a reversed-phase column (Vydac Everest 238EV5.07515, 75 mm 6 150 mm) fitted with a coated spray tip (FS360-50-5-CE; New Objective, Inc.). A nanoflow LC system (Dionex, Sunnyvale, CA) with autosampler, column switching device, loading pump, and nanoflow solvent delivery system was used. Elution solvents were A (0.5 acetic acid in water) and 
B (0.5 acetic acid in 80 acetonitrile/20 water). Samples were eluted at 250 nL/min using a binary gradient (8 B at 0 min to 80 B in a 30 min linear gradient, held at 80 B for 5 min, then back to 8 B for 15 minutes). The QStar Pulsar was externally calibrated daily with human [Glu1]-fibrinopeptide B. In parallel, 1 mL of the Gnd stock solution was mixed with with 49 mL of sinapinic acid (SA) solution (10 mg/mL SA dissolved in 30 ACN with 0.3 TFA) and analyzed by MALDI-TOF. One half mL aliquots were deposited using the dried-droplet method onto a 384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA). MALDI analysis was performed in a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems, Foster City, CA, USA). MS spectra were acquired in linear mode (20 kV source) with a Nd:YAG, (355 nm) laser, and averaging 500 laser shots. The mass of the peptide M153-S232 (9573 Da) was determined by an iterative calibration approach, using insulin (m/z = 5733), ribonuclease A (m/z = 13682) and lysozyme (m/ z = 14305), (Sigma-Aldrich, St. Louis, MO) as internal standards. Then, the signals from the M153-S232 (9573 Da), G89-S232 (16371 Da), and G81-S232 (17148 Da) peptides were used to calibrate the rest of peaks in the spectrum. Masses were matched to PrP fragments with the help of GPMAW 6.0 1326631 software (Lighthouse, Odense, Denmark).PK. Both samples were treated with PNGase F. WB was probed with the #51 antibody. (TIF)Figure S2 Characterization of isolated GPI2 PrPSc. 10 ml of sample were loaded and separated in a 15 gel by SDS-PAGE. The gel was stained by Coomassie blue. The molecular weight of the GPI-less PrP27-30 is ,16750 Da. (TIF) Figure S3 Western blot of PK-resistant fragments. In unpurified (1) and purified GPI- PrPSc (2). Both samples were digested with proteinase K, 25 mg/ml and 10 mg/ml, respectively, treated with PNGase F and resolved on a Tricine-SDS-PAGE gel. WB was probed with the R1 antibody. (TIF) Figure S4 Bayesian protein reconstruction of the nanoLC-ESI-MS spectra of PK-treated purified GPI2 PrPSc. The mass graphs of the three peaks: 17148 Da (top), 16729 Da (middle) and 16371 Da (bottom), identified by ESI-TOF are shown. (TIF) Figure S5 Western blot of recombinant MoPrP(23?31)cleavage by PK. Samples were digested with.
