The sheep PrP amino acid sequence was obtained from the UniProt database (accession number Q712V9 for sheep with VRQ/VRQ polymorphism). PeptideMass [40] was used to obtain theoretical masses of trypsin digested peptides. MaxQuant [41] was used to identify and quantify all the peptides present in the sample, with methionine oxidation and N-terminus acetylation as variable modifications, and cysteine carbamidomethylation as a fixed modification. The identification of glycopeptides was performed manually using DataAnalysis software (version 4.4, Bruker).

Extracted ion chromatogram (EIC) of the MS/MS data for the m/z value 366.13 (1+), representing [HexNAcHex + H]+ was created and each sample was searched for the presence of the glycan oxonium ions, such as the aforementioned, together with 204.08 (1+), representing [HexNAc + H]+ and in case of sialylation 292.09 (1+), representing [NeuAc + H]+. GlycoWorkbench [45] was used to display glycan structures, also calculating theoretical masses of the glycopeptide fragments, which were searched for in the MS/MS spectra. After confirming the presence of prion glycopeptides at a given retention time, the base peak chromatogram (BPC) was searched for other m/z values, and a list of all multiply charged ions was created, corresponding to potential prion glycopeptides. The values were converted to singly charged ions and characterized using GlycoMod [46], proposing all the possible glycan compositions. After determining all the possible glycopeptides, EIC was created for all the compositions, by including m/z values of all multiply charged states.

All EICs were integrated using DataAnalysis, and relative abundances were normalized by total area by dividing the peak area of each glycopeptide in each sample with the total chromatographic area of the sample.

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