Phosphopeptide Enrichment from HILIC Fractions

Dimethyl labeled (1:1 mixed conditions) from three biological replicates were fractionated using Ultimate 3000 HPLC (Thermo Scientific) equipped with a 4.6 × 250- mm TSKgel Amide-80 5-μm particle column (Tosoh Biosciences). The buffers used for the separation were 0.1% TFA (HILIC buffer A) and 99.9% acetonitrile, 0.1% TFA (HILIC buffer B). The peptide samples were resuspended in 80% HILIC buffer B and injected onto the HILIC column. The chromatography was performed using the following elution gradient: 80% B held for 20 min followed by 80% B to 60% B in 40 min and finally 0% B for 10 min at a flow rate of 0.4 mL/min. In total, 16 fractions were collected per biological replicate and subjected to phosphopeptide enrichment using titanium dioxide (TiO₂, Titansphere, GL Science) as previously described [20 (link),23 (link)]. Briefly, TiO₂ spheres were prepared at 50 mg/mL, and 25 μL (1.25 mg TiO2) were added to each sample fraction, incubated for 10 min/RT to bind phosphopeptides. Samples were washed and eluted with 200 μL 0.4 M NH₄OH followed with 200 μL 0.2 M NH₄OH/50% acetonitrile and then dried using a speedvac (Genevac).