DOI: 10.25345/c50m0q
title: MassIVE MSV000084018 - An Induced-Fit Asymmetric FGF Receptor Kinase Dimer Mediates A-Loop Tyrosine Transphosphorylation
title: The data are the result of LC-MS/MS (Thermo Fisher Q Exactive HF) analysis of a tryptic digest of the his-tagged FGFR2 kinase domain after excision of the appropriate band from an SDS-PAGE gel. Each dataset result from analysis of either WT kinase domain or various forms harboring the indicated amino acid substitutions, after the indicated time of incubation under trans-phosphorylating conditions. Trans-phosphorylation on A-loop tyrosines was initiated by mixing wild-type or mutated FGFR kinases with reaction buffer containing ATP and MgCl2 to final concentrations of 67.5 microM (kinase), 25 mM (ATP) and 50 mM MgCl2.  Reactions were quenched at different times by adding EDTA (final concentration, 50 mM) to the reaction mixture. kinase reaction products were resolved by SDS-PAGE (14%), the proteins stained with Bio-safe Coomassie G250 (Bio-Rad), and bands were excised.  Following complete destaining with 50% methanol and 25mM NH4HCO3 / 30% acetonitrile, each gel section was diced into small pieces, dehydrated with acetonitrile and dried by vacuum centrifugation.  Gel pieces were rehydrated with 12.5 ng/microliter protease trypsin solution (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI, USA) in 50 mM NH4HCO3 and incubated at 37degree C for 4 hours.  The resulting peptides were extracted twice with 5% formic acid / 50% acetonitrile followed by a final extraction with acetonitrile.  Samples were concentrated by vacuum centrifugation and peptides were desalted using a Stage Tip manually packed with Empora C18 High Performance Extraction Disks (3M, St. Paul, MN, USA, [Rappsilber J, 2010]).  LC-MS/MS analysis was performed using a Thermo Scientific Q Exactive High Field (QE HF) mass spectrometer coupled to a Thermo Scientific EASY-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a self-packed 75micrometer x 20-cm reverse phase column (New Objective PicoTip Emitter; New Objective, Woburn, MA, USA) packed with Reprosil C18, 3 microm, Dr. Maisch GmbH, Germany) for peptide separation.  The analytical column was placed in a column heater (Sonation GmbH, Biberach, Germany) set to 45 degree C.  Peptide mixtures were loaded onto the analytical column with buffer A (0.1% formic acid) at a maximum back-pressure of 300 bar; they were then eluted with a 3-40% acetonitrile gradient in 0.1% formic acid over 60 min at a flow rate of 250 nL/min.  The mass spectrometer was operated in data-dependent (DDA) mode with survey scans acquired at a resolution of 120,000 over a scan range of 300-1750 m/z.  Up to ten of the most abundant precursors from the survey scan were selected with an isolation window of 1.6Th and fragmented by higher-energy collisional dissociation with a normalized collision energy (NCE) of 27.  The maximum ion injection time for the survey and MS/MS scans was 60ms and the ion target value for both scan modes was set to 3e (Rappsilber et al., 2007). Trans-phosphorylation on A-loop tyrosines of catalytically inactive kinase substrates (i.e. WTCI, R678GCI and R678ECI) was initiated by mixing WT FGFR2 kinase with dead substrates in reaction buffer to final concentrations of 13.5 microM (enzyme), 67.5 microM (dead substrates), 25 mM (ATP) and 50 mM (MgCl2).  Subsequent quantitation of the kinetics of A-loop trans-phosphorylation for catalytically dead substrates were done using LC-MS as described above. The methods and results are described in a manuscript by Lingfeng Chen, William M. Marsiglia, Huaibin Chen, Joseph Katigbak, Hediye Erdjument-Bromage, David J. Kemble, Lili Fu, Jinghong Ma, Gongqin Sun, Yingkai Zhang, Guang Liang, Thomas A. Neubert, Xiaokun Li, Nathaniel J. Traaseth, and Moosa Mohammadi entitled An Induced-Fit Asymmetric FGF Receptor Kinase Dimer Mediates A-Loop Tyrosine Transphosphorylation.