Figure 4.

Improved phosphopeptide enrichment protocols with TiO₂ using glycolic acid. (A) Profiles of the number of phosphopeptides identified and phosphopeptide enrichment specificity with the four modified phosphopeptide enrichment protocols that used glycolic acid. Three replicates are performed for each protocol. “Normal” represents the normal phosphopeptide enrichment protocol using glycolic acid; “PNGaseF” represents that PNGaseF is used to remove glycans from peptides before TiO₂ enrichment; “2X washings” represents that the volume of the loading and washing buffers is double that of the normal phosphopeptide enrichment protocol; “Changed washing buffer” represents that different washing buffers are used during phosphopeptide enrichment; “Double_TiO₂” represents that two rounds of TiO₂ enrichment are performed for each experiment. (B) Overlap between phosphopeptides identified in TiO₂-TiO₂ (double TiO₂ enrichment) and TiO₂-TiO₂-FT (the supernatant of double TiO₂ enrichment). The combined results of three replicates are shown. (C) The percentage of singly phosphorylated, doubly phosphorylated, and triply phosphorylated peptides in TiO₂ enrichment (normal phosphopeptide enrichment protocol using glycolic acid), TiO₂-TiO₂ enrichment, and TiO₂-TiO₂-FT. Bars show mean ± SD of three replicates; *** p < 0.001 (one-way ANOVA with LSD post hoc test). (D) The percentage of phosphorylated amino acids (serine(pS), threonine (pT), and tyrosine (pY)) in TiO₂ enrichment, TiO₂-TiO₂ enrichment, and TiO₂-TiO₂-FT. Bars show mean ± SD of the three replicates; * p < 0.05, *** p < 0.001 (one-way ANOVA with LSD post hoc test).