Ot (Figure S1). Female mice were intracerebrally inoculated at six weeks of age with 20 ml of a 2 RML-infected mouse brain homogenate (BH), ?kindly provided by Juan Maria Torres, CISA, Madrid, Spain. After 365 days post inoculation, the asymptomatic mice [16] were euthanized, their brains surgically removed, 223488-57-1 site rinsed in PBS, and stored at 280uC until needed.Structural Organization of Mammalian Prionswere used as a secondary antibody, as appropriate (1:5000 dilution). Blots were developed with ECL-plus reagent 25033180 (GE Healthcare, Little Chalfont, UK). Three sets of partially overlapping MW markers, Peptide Molecular 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 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 MedChemExpress PHCCC 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 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) Fig.Ot (Figure S1). Female mice were intracerebrally inoculated at six weeks of age with 20 ml of a 2 RML-infected mouse brain homogenate (BH), ?kindly provided by Juan Maria Torres, CISA, Madrid, Spain. After 365 days post inoculation, the asymptomatic mice [16] were euthanized, their brains surgically removed, rinsed in PBS, and stored at 280uC until needed.Structural Organization of Mammalian Prionswere used as a secondary antibody, as appropriate (1:5000 dilution). Blots were developed with ECL-plus reagent 25033180 (GE Healthcare, Little Chalfont, UK). Three sets of partially overlapping MW markers, Peptide Molecular 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 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 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) Fig.
