Bond elut omix c18 pipette tips

Dried peptide samples (~380 µg) were sonicated in 50 µl of solution 0 (2.5% acetonitrile, 0.5% TFA) and 100 µl of solution 2 (80% acetonitrile, 0.5% TFA, 100% lactic acid). Titansphere Phos-TiO Kit spin tip-columns (GL Sciences, Tokyo, Japan) were washed with 40 µl of solution 1 (80% acetonitrile, 0.5% TFA). Samples were loaded on the spin tip-columns and passaged three times through a centrifuge; 5 min at 200 g, 15 min incubation at room temperature, and 10 min at 200 g. Spin tip-columns were subsequently washed once with solution 1, twice with solution 2, and twice with solution 1 for 2 min at 200 g. Phosphopeptides were eluted in two steps, first with 50 µl of 5% ammonium hydroxide (5 min at 200 g) and secondly with 5% pyrrolidine solution. A 20 µl aliquot of 20% formic acid was added to lower the pH, and samples were cleaned on Bond Elut OMIX C18 pipette tips (Agilent Technologies) following the manufacturer’s instruction.