Bridged ethyl hybrid c18 analytical column

Phosphorylation sites of talin-1 R7R8 were determined using mass spectrometry. The in vitro kinase assay was carried out with the substrate talin-1 R7R8 and CDK1-cyclin A2. Reactions was carried out for 45 min at 30 °C and stopped by adding SDS sample buffer and boiling for 10 min at 95 °C. All samples were loaded onto an SDS PAGE 4 to 12% Bis-Tris gel (Thermo Fisher Scientific) and separated by running at 200 V for 60 min. Gels were stained with Instant-Blue (Expedeon) for 15 min and washed in water overnight at 4 °C. The talin R7R8 bands were cut from the gel and processed by in-gel tryptic digestion. Peptides were analyzed by LC-MS/MS by using an UltiMate 3000 Rapid Separation LC (Dionex Corporation) coupled to an Orbitrap Elite MS (Thermo Fisher Scientific). Peptides were separated on a bridged ethyl hybrid C18 analytical column (250 mm × 75 μm internal diameter, 1.7 μm particle size; Waters) over a 45 min gradient from 8 to 33% (v/v) acetonitrile in 0.1% (v/v) formic acid. LC-MS/MS analyses were operated in data-dependent mode to automatically select peptides for fragmentation by CID. Multistage activation was enabled to fragment product ions resulting from neutral loss of phosphoric acid. Quantification was performed using Progenesis LC-MS/MS software.