FLNc Calpain GluC SRM Analysis
Lena Reimann, Heike Wiese, Yvonne Leber, Anja N Schwäble, Anna L. Fricke, Anne Rohland, Bettina Knapp, Christian D. Peikert, Friedel Drepper, Peter F.M. van der Ven, Gerald Radziwill, Dieter O Fürst and Bettina Warscheid
- Organism: Human
- Instrument: QExactive Plus
Human filamin C (FLNc) is a target of the protease calpain at Y2625. To confirm the dependency of calpain cleavage from FLNc phosphorylation at position S2623/S2624 by PKCa, FLNc was overexpressed in HEK293 cells and cells were treated with PMA to stimulated kinase activity, with Gö6976 to inhibit kinase activity or mock-treated with DMSO. Subsequently, recombinant calpain 1 and GluC were used and the resulting peptide TVTSSSSRGSSY was monitored by SRM analyses on a QExactive Plus.
For calpainolysis assays, HEK293 cells transiently expressing Myc-FLNc d22-24 WT were used. Prior to lysis, cells were treated with PMA (200 nM for 15 min) or Gö6976 (10 uM for 1 h). Cell lysis was performed 24 h after transfection on ice with pre-cooled lysis buffer (10 mM Tris HCl, pH 7.4, 100 mM NaCl, 0.1% Triton X 100, 2 mM EGTA, sodium fluoride, sodium pyrophosphate, sodium orthovanadate, beta-glycerophosphate). Subsequent to lysis, cells were incubated in an ultrasonic bath for 20 min at 8°C and centrifuged for 5 min at 1,500 x g. Supernatants were adjusted to the same protein concentration and calpainolysis was started by addition of 5 mM CaCl2 and 0.85 ug calpain 1 (Merck) per 100 ul reaction volume. Samples were incubated for 20 min at 37°C and 800 rpm. Following the digestion, N-terminal FLNc was enriched by Myc-directed immunoprecipitation. Myc-bound protein was digested on bead with GluC (1:50) for 4 h at 42°C. The resulting cleavage peptide TVTSSSSRGSSY was anlysed by SRM analyses on an QExactive Plus with a dynamic exclusion of 3 seconds, a max ion time of 200 ms a loop count of 5 with an MSX count of 1 and a isolation window of 4 m/z. Resulting Raw files were searched with MaxQuant 126.96.36.199 against the Uniprot Proteomest database (release 01.12.2015, 57,276 protein entries). The precursor mass tolerance was set to 20 ppm for the first search and to 4.5 ppm for the main search. GluC and calpain 1 were set as proteolytic enzyme allowing up to one missed cleavage. Resulting msms.txt file was imported into Skyline and peak intensities were manually corrected and normalized to the total MaxQuant TIC intensity of the corresponding run.
Created on 10/7/16, 9:24 AM