Monocap c18 high resolution 2000 column

Desalted phosphopeptides were analyzed in the TripleTOF5600 system (AB SCIEX, Framingham, MA, USA) equipped with Autosampler-2 1D plus (Eksigent, Framingham, MA, USA) and NanoLC Ultra (Eksigent, Framingham, MA, USA) using a MonoCap C18 High-Resolution 2000 column (GL Sciences Inc., Tokyo, Japan) and PicoTip emitter SilicaTip (New Objective Inc., Woburn, MA, USA). Peptides were eluted at a flow rate of 500 nL/min⁻¹ with a four-step gradient using 0.5% (v/v) acetic acid: 0.5% (v/v) and 80% (v/v) acetic acid = 98:2 (0 min), 60:40 (300 min), 10:90 (20 min), and 98:2 (40 min). The eluate was sprayed into the mass spectrometer by electrospray ionization (ESI). The mass spectrometry (MS) scan range was 400–1250 m/z, and the MS/MS scan range was 100–1600 m/z.

Mass spectrometric analysis was performed using a TripleTOF 5600 instrument (SCIEX) with an Autosampler-2 1D Plus and NanoLC Ultra (Eksigent). Each sample was isolated using a MonoCap C18 High-Resolution 2000 column (2000 mm × 100-μm inside diameter, 2-μm pore size; GL Science, Japan). Four microliters of the sample were concentrated through the analytical column at a flow rate of 500 nL/min for 30 min. The mobile phase comprised of 2% acetonitrile and 0.1% formic acid (A) and 80% acetonitrile and 0.1% formic acid (B). The following linear gradient was used in this analysis: A:B = 98:2 at 0 min to A:B = 60:40 over 300 min, A:B = 10:90 over 20 min and A:B = 98:2 over 40 min. The MS scan range was a mass/charge ratio (m/z) of 400 to 1250, and the top 20 precursor ions were selected for subsequent MS/MS scans in the high-sensitivity mode. The MS/MS data were analysed using ProteinPilot 5.0 software (SCIEX) and subsequently annotated using the A. thaliana TAIR10 protein database for peptide identification.