Ltq orbitrap xl lc ms ms system

The in vitro phosphorylation for MS analysis was performed in a 20 μl reaction for 2 hr at room temperature. The reaction buffer contains 20 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 5 mM EDTA, 1 mM DTT, 5 mM ATP, 10 μg of GST-BTL2^JK with or without 1 μg of MBP-BAK1^JK. The phosphorylated GST-BTL2^JK proteins were resolved by 10% SDS-PAGE gel. The gel was stained with Thermo GelCode Blue Safe Protein Stain and destained with ddH₂O. The corresponding bands were sliced and subjected to in-gel digestion with trypsin. The phospho-peptides were enriched and analyzed using an LTQ Orbitrap XL LC-MS/MS system (Thermo Scientific) as previously described^{78 (link)}. The MS/MS spectra were analyzed with Mascot (Matrix Science; version 2.2.2), and the identified phosphorylated peptides were manually inspected to ensure confidence in phosphorylation site assignment.