Ultimate 3000 nano lc hplc system

In order to analyse Thr85 autophosphorylation in the G87R mutant, 1 μg of WT CaMKK2 or G87R mutant was incubated with 5 mM MgCl₂, 50 μM CaCl₂ and 1 μM CaM in the presence and absence of 200 μM ATP for 40 min, then digested with trypsin and analyzed by reversed-phase HPLC-ESI-MS/MS using an UltiMate 3000 Nano LC HPLC system (Dionex) directly connected to a Triple-TOF 5600 mass spectrometer (AB SCIEX) in direct injection mode. Peptide mixtures were resolved on an analytical nanocapillary HPLC column (100 μm i.d. × 15 cm) packed with C₁₈ Acclaim PepMap100 (3 μm particle size, 100 Å pore size) using a 1–75% elution gradient of 98% acetonitrile/2% of 0.1% formic acid (v/v) in water at a flow rate of 250 nl/min. Mass spectrometric data were analyzed using the database search engine ProteinPilot and the Paragon algorithm (AB Sciex).

For phosphopeptide analysis, 1 μg of CaMKK2 was digested with trypsin and analyzed by reversed-phase nHPLC-ESI-MS/MS using an UltiMate 3000 Nano LC HPLC system (Dionex) directly connected to a Triple-TOF 5600 mass spectrometer (AB SCIEX) in direct injection mode. Peptide mixtures were resolved on an analytical nanocapillary HPLC column (100 μm i.d. × 15 cm) packed with C₁₈ Acclaim PepMap100 (3 μm particle size, 100 Å pore size) using a 1–75% elution gradient of 98% acetonitrile/2% of 0.1% formic acid (v/v) in water at a flow rate of 250 nl/min. Mass spectrometric data were analyzed using the database search engine ProteinPilot and the Paragon algorithm.