All frozen and fixed brain tissues were obtained from the National Prion Disease Pathology Surveillance Center (NPDPSC) at Case Western Reserve University (CWRU) in Cleveland, OH. A cohort of 23 subjects was selected that, according to common sCJD subtype classifications, comprised 21 cases harboring exclusively one histotype, and 2 cases with mixtures of two co-existing histotypes [1, 27]. The 21 sCJD “pure” cases cohort included: 4 MM1; 3 MV1; 4 MM2; 4 MV2C; 3 MV2K; 3 VV2. Three cases of pure MV2C, MV2K, and MV1 each and two cases in which the histotype was mixed were used for mass spectrometry analyses. In one of the mixed cases, the MV2K and MV2C histotypes co-existed in equal ratio (50:50); in the other one, the cerebral cortex showed pure MV2C histotype while the mixed MV2K + C histotype with a 90:10 ratio was observed in the cerebellar cortex.
Reagents and antibodies
Tris-buffered saline (TBS), Tris/glycine buffer, Tris/glycine/SDS buffer, Tween 20, β–mercaptoethanol, Dithiothreitol (DTT), Laemmli sample buffer, Non-fat dry milk, 15% Criterion Tris-HCl polyacrylamide gels were procured from Bio-Rad Laboratories (Hercules, CA, USA). Benzonase, Polyvinylidene difluoride (PVDF) (Immobilion-P and Immobilon-FL) were purchased from Millipore Sigma (Burlington, MA, USA). 1 M Tris-HCl, N-Lauroylsarcosine sodium salt solution, 3- Indole acetic acid, 8 M Guanidine-hydrochloride, (GdnHCl), Calcium chloride, Phenylmethylsulfonyl fluoride (PMSF), Dulbecco’s Phosphate Buffered Saline (D-PBS), Proteinase K, Iodoacetamide (IAA) and Ammonium bicarbonate were obtained from Sigma-Aldrich. NuPAGE 4–12% Bis-Tris gradient gels, NuPAGE MES SDS running buffer (20X), Acetonitrile, Silver Staining kit, Ethanol, Formic Acid, Methanol, Pierce ECL 2 Western Blotting Substrate, Odyssey Blocking Buffer and Trypsin from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The following antibodies were used: 3F4 (to human PrP residues 106–110) , 12B2 (to human PrP residues 89–93) [18, 32], Tohoku-2, polyclonal antibody (to residues 97–103)  and SAF32 (to human prion octapeptide repeat region 59–89) , Alkaline phosphatase-conjugated goat anti mouse or anti rabbit (Promega, Madison, WI, US), Horseradish peroxidase (HRP)- conjugated sheep anti-mouse or donkey anti-rabbit IgG Ab sheep anti-mouse IgG (GE Healthcare Life Sciences, Chicago, IL, USA) and Infrared Dye (IRDye) 800CW goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA).
DNA was isolated from frozen brain tissues and genotyping of PRNP coding region was performed as previously described .
Histopathology and immunohistochemistry were performed essentially as described previously [5, 6].
PK-resistant PrPD analysis
Brain homogenates (BH) (10% w/v) were prepared as previously described . Brain homogenate was centrifuged at 1000 x g for 10 min and supernatant (S1) was collected. S1 were treated with 70 U/ml proteinase K (PK) and incubated at 37 °C for 1 h. Protease digestion was stopped by adding 4 mM PMSF. Samples were mixed with equal volumes of 2X sample buffer (6% SDS, 8% 2-mercaptoethanol, 20% glycerol, 4 mM EDTA, 125 mM Tris HCl pH 6.8), boiled for 10 min and incubated with 8-fold pre-chilled methanol overnight at − 20 °C. The following day, the samples were centrifuged at 18,200 x g for 30 min, pellets were resuspended in 1X sample buffer by sonication and subjected to immunoblotting.
Protein samples (brain tissue equivalent 0.1–3 mg of wet tissue) were separated in 4–12% NuPAGE Bis-Tris gradient gels and blotted into Immobilon-FL membranes for 40 min at 30 V, blocked with 5% nonfat dry milk in TBS-T (1× TBS with 0.1% Tween 20) and probed with the indicated antibodies. Immunoblots were visualized by ImageQuant LAS 4000 or by Odyssey infrared imaging system (LICOR Biosciences) as described by the manufacturer.
Conformational stability immunoassay (CSI)
CSI was performed according to previous protocols [4, 30, 35] with slight modifications. Aliquots of BH (10% w/v) prepared in LB100 (pH 8.0) were centrifuged at 1000 × g for 10 min at 4 °C and the S1 fraction were collected. Fifty μl aliquot of S1 was diluted with an equal volume of GdnHCl to obtain final GdnHCl concentrations ranging from 0 to 3.5 M and samples were incubated for 1.5 h at 22 °C. GdnHCl was subsequently removed by incubating each sample with an excess of 8-fold pre-chilled methanol overnight at − 20 °C, followed by centrifugation at 18,200 × g for 30 min. Pellets were resuspended in 50 μl LB100 (pH 8.0) by sonication. Each aliquot was digested with 10 U/ml PK for 1 h at 37 °C and reaction was terminated by addition of 4 mM PMSF. Samples were denatured and loaded onto 15% precast Tris-HCl gels. PrP amounts at different GdnHCl concentrations were measured by Odyssey application software V3.0. The conformational stability curves (i.e., fraction of remaining PrPD as a function of GdnHCl concentration) for unglycosylated PrPD were fitted to a sigmoidal equation using GraphPad Prism. The mean [GdnHCl]1/2 values ± standard error of the mean (SEM) were calculated and compared between histotype case groups.
Proteinase K titration assay
The proteinase K (PK) titration assay was performed as described previously . Briefly, 30 μl of S1 was treated with different concentrations of PK (0.62, 1.25, 2.5, 5, 10, 20, 40, 80, 160 U/ml) for 1 h at 37 °C. Additionally, S1 was incubated with 320 U/ml of PK for 12 h at 37 °C. The PK digestion was terminated with 4 mM PMSF; equal aliquots of 2X sample buffer were added and samples were boiled for 10 min and subjected to immunoblotting with 3F4. The PK points from 5 to 320 U/ml of the unglycosylated resPrPD fragment from sCJD variants were best fitted using one phase decay and PK½ index values ± SEM were determined and compared between subtypes.
Purification of resPrPD
Purification of resPrPD was performed as previously reported  following extraction from the cerebral cortex (CC) of sCJDMV1, MM2, MV2C, MV2K, VV2 and and MV2K-C cases. In a mixed case containing the sCJDMV2C histotype in the cerebral cortex and the sCJDMV2K in the cerebellum, resPrPD was purified also from the cerebellar cortex.
Purity of resPrPD corresponding to different sCJD subtypes was analyzed by gel electrophoresis with silver staining detection according to manufacturer instructions (Thermo Fisher Scientific Inc., Waltham, MA, USA).
In-solution digestion of purified resPrPD
In-solution trypsin digestion was performed according to the previously method  with some modifications. Briefly, purified resPrPD samples were dissociated by incubation with 8 M GdnHCl for 10 min; this was followed by reduction with DTT and alkylation with IAA step. After centrifugation, the supernatant was collected and incubated overnight at − 20 °C with nine volumes of methanol to precipitate the protein. The precipitates were pelleted by centrifugation at 21,130 x g for 30 min at 4 °C and then resuspended in 100 mM ammonium bicarbonate. Trypsin (MS grade, Thermo Scientific) was dissolved according to the manufacturer’s instructions and added to the protein solution at the enzyme to resPrPD weight ratio of 1:10. The mixture was incubated overnight at 37 °C with shaking, and the reaction was terminated by adding formic acid to a final concentration of 5% (vol/vol). Samples were concentrated to a volume of ~ 20 μl and stored at − 80 °C until analysis by Nano LC-MS.
Nanospray LC-MS-MS analysis was performed using an LTQ Orbitrap XL mass spectrometer equipped with nanoelectrospray source (Thermo Scientific, San Jose, CA). Trypsin-digested samples were loaded onto a C-18 trap column (to remove salts) and separated on a C-18 column (Acclaim PepMap, Thermo Scientific, CA) connected to an emitter. Separation was performed using a Dionex UltiMate 3000 system (Thermo Scientific, San Jose, CA) and a gradient of acetonitrile in water containing 0.1% formic acid. The flow rate was 300 nl/min. The mass spectrometer was externally calibrated using a Pierce LTQ ESI positive ion calibration solution (Thermo Scientific, catalog number 88322). MS-MS data in full scan experiments were acquired in the m/z 300–1800 range at a resolution of 30,000 (FWHM at m/z 400). The following source settings were used: spray voltage = 4.2 kV; capillary temperature = 200 °C. Data-dependent MSn (n = 2) were acquired at ITMS using collision induced dissociation (CID); the top 14 intense ions were subjected for further fragmentation. Calculation of elemental formulae was performed on the mono-isotopic peak of each ion cluster using Xcalibur software v2.2 with a mass tolerance of 3 to 5 ppm. MS/MS raw files were searched using MASCOT Deamon engine against the database containing sequence of human prion protein 129 M/V. Trypsin/P search parameters for Mascot peptide identification included one missed tryptic cleavage, fixed carbamidomethylation (+ 57 Da, Cys), and variable oxidation (+ 16 Da, Met). Mass tolerances of 2.0 and 1.0 Da were used for parent and monoisotopic fragment ions, respectively. The resulting files generated by MASCOT were used for peptide identification with the constraints that only MASCOT ion scores greater than 10 were considered. The percentage of 129 M and 129 V PrP in resPrPD samples was calculated by the spectral counting method .
Calibration curves for allotypic ratio determination
To obtain a calibration curve, recombinant full-length human 129 M and 129 V PrP were purified as described previously [21, 33] and the concentration of each protein was determined by absorbance at 280 nm using the extinction coefficient of 57,995 M− 1 cm− 1. Both proteins were combined at the 129 M PrP to 129 V PrP molar ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. After in-solution trypsin digestion, the samples were analyzed by mass spectrometry to determine the relative ratio of the 129 M and 129 V polymorphs as described above. A similar calibration curve was also obtained using purified human resPrPD from sCJDMM2 and sCJDVV2 subtypes by combining the proteins at the same the molar ratios. For this purpose, resPrPD from sCJDMM2 and sCJDVV2 subtypes were quantified from Western blots by densitometry using the Odyssey application software V3.0. Recombinant PrP was used as a standard for this Western blot-based quantification.
Statistical significance was assessed with the one-way analysis of variance (ANOVA) followed by multiple pairwise comparisons with the Tukey test supported by GraphPad Prism 8.0.