Fig. 3

Two-step enrichment strategy and the optimization of the two-step enrichment experiment conditions. a The experimental strategy for pY39 αβ-syn peptide enrichment using the two-step enrichment approach in which phosphotyrosine peptides were enriched followed by total phosphopeptides were enriched using TiO₂ beads. CSF proteins were digested with Lys-C and trypsin. To monitor the enrichment efficiency, heavy standard pY39 αβ-syn peptide was added before the two-step enrichment and the enrichments were conducted. The endogenous light and heavy standard pY39 αSyn were monitored under PRM mode followed by quantification using Skyline software. b Different volumes of agarose beads coupled with anti-phosphotyrosine antibodies were incubated with the target peptides to investigate the best ratio of anti-phosphotyrosine agarose beads to the target peptides. c Different amounts of TiO₂ beads were incubated with the target peptides to investigate the best ratio of TiO₂ beads to the target peptides