AKT2 interacting proteins_ MBP- and MAP-TAPs

Tagged AKT2 was expressed in HEK293T cells. For quantification SILAC labeling was performed. MBP-TAP: Cell lysates were mixed at a 1:1 ratio with unlabeled wild type cells before Tandem Affinity Purification. MAP-TAP: Same amount of labeled and unlabeled cell lysates were purified via Tandem Affinity Purification and afterwards eluates were mixed at a 1:1 ratio. AKT2 and co-purified proteins were digested with trypsin, fractionated via SCX and analyzed via LC-MS. Sequence database-search of the MS data was performed against the uniprot human taxonomy-9606 database containing 83659 entries using the SEQUEST algorithm with the following parameters: trypsin specificity, two missed cleavage sites, precurser ion mass accuracy tolerance of 10–30 ppm, cysteine carbamidomethylation, methionine oxidation, pSTY, N-terminal protein acetylation and, when performed, SILAC labels Lys-6, Arg-6 specified as modifications. The minimal cross-correlation score (XCorr) was set to 2.0, 2.5 and 3.0 for charge states +2, +3 and +4 respectively. The Delta Cn had to be >0.1 and the minimal peptide probability allowed was 0.05. The minimum number of peptides necessary for protein identification was three.