Easy nano lc instrument

ESI-MS was performed on the purified enzymes as described previously (58 (link)). In short, a buffer exchange to 0.1% formic acid (Merck Millipore, Burlington, MA, USA) was performed using centrifugal molecular cutoff filters (Merck Millipore; 10,000 Da). The protein masses were determined using an Orbitrap Fusion Lumos device (Thermo Fisher Scientific). Injection was performed using an EASY-nano LC instrument (Thermo Fisher Scientific) with a 15-cm C₁₈ EASY-spray column. Mass calculations were done using BioPharma Finder 3.0 protein deconvolution software (Thermo Fisher Scientific, Massachusetts, USA).

Lysates (5 mg) derived from HEK-293 cells stably expressing wild-type or mutant FLAG epitope-tagged KCC3 were subjected to immunoprecipitation with anti-FLAG antibody covalently conjugated to agarose (5 μl). Immunoprecipitates were washed three times with lysis buffer containing 0.5 M NaCl, followed by two washes with Buffer A. Proteins were eluted from FLAG beads by resuspendion of immunoprecipitates in SDS sample buffer (30 μl). The immunoprecipitates were subjected to electrophoresis on a precast 4–12% gradient gel (Invitrogen) and the protein bands were visualized following Colloidal Blue staining. Proteins in the selected gel bands were reduced and alkylated by the addition of 10 mM DTT, followed by 50 mM iodoacetamide. Identification of proteins was performed by in-gel digestion of the proteins with 5 μg/ml trypsin and subsequent analysis of the tryptic peptides by LC (liquid chromatography)–MS/MS (tandem MS) on a Thermo LTQ-Orbitrap system coupled to a Thermo Easy nano-LC instrument. Excalibur RAW files were converted into peak lists by Raw2msm70 (link) and then analysed by Mascot (http://www.matrixscience.com), utilizing the SwissProt human database. Two missed cleavages were permitted; the significance threshold was P < 0.05.