Kinetex 5u evo c18 100a

A peptide‐fragment library was built in‐house to match against the acquired DIA spectra. Therefore, 10 μg of each sample was pooled for the full peptide library and 20 μg of each sample was pooled for the phosphopeptide library. Pooled samples were fractionated on a high‐pH reversed‐phase C18 column (Kinetex 5u Evo C18 100A, 150 × 2.1 mm; Phenomenex) coupled to an Agilent 1100 series HPLC over a 50 min gradient. Fractions were concatenated to 20 fractions for the proteome library and seven fractions for the phosphoproteome library. Samples were dried down and stored at −20 °C. Full peptide library samples were read for MS analysis. The phospho‐peptide library samples were enriched for phosphorylated peptides as described previously [14 (link)].

For analytical HPLC, the dry crude extract was resuspended in methanol (MeOH) and filtered through a 0.45 μm syringe filter (Merck, Darmstadt, Germany) and 20 μL of the filtered extract was injected. Sample profiles were obtained from an UltiMate 3000 HPLC system coupled with WPS-3000(T) Autosampler, HPG-3400 pump, and DAD-300 detector. The separation was performed on a Raptor ARC-18 column, 2.7 μm, 150 × 4.6 mm (Restek, 9314A65). Diluent: 25:75 water: methanol, Inj. Vol: 5 μL. Mobile phase: A: water, 5 mM ammonium formate, 0.1% formic acid; B: acetonitrile, 0.1% formic acid. Isocratic (%B): 0 min (75%), 9 min (75%) at a flow rate of 1.5 mL min^-1. The compound peaks were detected at 220 and 280 nm.
For preparative HPLC 50 mg of the dry crude extract was dissolved in 10 mL solvent (75% MeOH and 25% water containing 0.1% acetic acid), and filtered through 0.45 μm syringe filter. Then, 10 mL of the filtered extract were injected. Sample separation in preparative HPLC was carried out using a Agilent Technologies 1260 Infinity preparative HPLC system, 1260 MWD-VL detector, Column: Kinetex 5u EVO C18 100A, 250 × 21.2 mm (Phenomenex). Mobile phase: A: water, 0.1% acetic acid, B: methanol. Gradient: (%B): 0.00 min (60%), 45 min (85%) for total run of 55 min.