Human Proteome Dataset Hek293, k652 and HeLa cell lines
Triple Silac approach to analyze cellline specific protein expression. Hek293 (Heavy, Lys8 and Arg10), K526 (Medium, DMEM with Lys 4 and Arg6), HeLa (Light).
Sample Processing Protocol
K562 cells were cultivated in RPMI-1640 medium supplemented with 10% FBS, 1% P/S, 1% gentamicin and 25 mM HEPES. HeLa and HEK293 cells were grown in DMEM supplemented with 10% dFBS, 1% P/S, 4mM glutamine, 84mg/ml Arg 6 and 146mg/ml Lys 4 or 84mg/ml Arg 10 and 146mg/ml Lys 8, respectively. Full incorporation of SILAC amino acids was confirmed by mass spectrometry. Subsequently, the cells were collected, lysed and the protein amount was determined by protein quantification assay (DC™ Protein Assay, BIO-RAD). This allowed mixing of the cells in order to obtain 1:1:1 protein ratio after the cell lysis. Larger protein complexes were precipitated with 0.25% v/v acetic acid and the samples were passed through 10kDa Modified PES Centrifugal Filter (VWR). Both supernatant and flow-thorough were digested with trypsin and analyzed by mass spectrometry.
Data Processing Protocol
MaxQuant was used for analyzing the dataset, using a Protein FDR of 1. The aim of the dataset was to look for evidences of short expressed proteins (micropeptides), therefore the Protein FDR was neglected. Apart from that standard MQ settings were applied.