The protocol was performed as described previously [85]. Briefly, bands corresponding to diglycosylated PrPSc were excised with a clean scalpel and cut into smaller cubes. The gel pieces were transferred to 1.5 mL microcentrifuge tubes and reduced with 10 mM of dithiothreitol (DTT, Sigma) for 30 min at 56°C. After cooling down, samples were alkylated with 55 mM iodoacetamide (IAA, Sigma) for 20 min at room temperature. Gel pieces were destained by alternating incubation with 100 mM ammonium bicarbonate/ACN (1:1, v/v) and neat ACN. Samples were digested with 6.5 ng/μL of trypsin (Promega) overnight at 37°C. Tryptic digests were extracted by shaking in 5% formic acid/ACN (1:2, v/v). Extraction was repeated two times, and the digests were pooled and dried in a vacuum concentrator.

Glycopeptide enrichment was performed using solid-phase extraction on Chromabond HILIC beads (Macherey-Nagel). The beads were added to wells of a filter plate (Orochem), the samples were loaded onto beads in 80% ACN containing 0.1% TFA (v/v) and washed two times with the same solvent. All the washes were removed by vacuum filtration (Pall). Elution was performed with 0.1% TFA, after which the glycopeptides were dried in a vacuum concentrator and reconstituted in 20 μL of ultrapure water.

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