AKT isoform specific signaling

Phosphoproteome analysis of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform specific knock down, respectively. Six biological replicates were analysed. Cells were grown until reaching 90 % confluency, starved for 50 minutes followed by stimulation with 200 nM insulin for 10 minutes. In solution stable isotope dimethyl labeling was performed following the protocol of Boersema et al. (2009) Nat. Protoc. 4, 484-494 Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific) was used according to the manufacturer’s instructions. Peptides were desalted with Graphite Spin Columns (Thermo Scientific) and fractionated by automated off-line 2-D LC. First dimension: 1 mm × 15 cm Polysulfoethyl-Aspartamide column (Dionex, Thermo Scientific). Two replicates were alternatively fractionated in-solution using the Agilent 3100 OFFGEL Fractionator (24 cm IPG strips pH 3-10 (GE Healthcare) and a 24 well frame set from Agilent Technologies). Peptides from the single SCX fractions were further separated and analyzed by reversed-phase nano-LC-MS/MS. Sequence database-search of the MS data was performed against the uniprot mouse database (downloaded 13.06.2012) using MaxQuant (Version 1.3.0.5).