The methodology of the experiment was based on the stable isotope labeling by amino acids in cell culture (SILAC) experiment whereby the Iscove’s modified minimal medium (IMEM) was custom prepared based on the ATCC formula without L-lysine or L-arginine. However, the complete light and heavy IMEM media was prepared in the addition of light and heavy lysine and arginine along in dialyzed FBS. The collected HL-60 cells were cultured in heavy IMEM medium for five cell doublings to achieve a complete isotope incorporation. The cultured medium was supplemented with 10% of fetal bovine serum and penicillin as antibiotics. The cultured cells were kept in a humidified atmosphere in presence of 5% CO2 at 370C. The medium used were renewed every two to three days depending on the cell density. Moreover, the SILAC experiment was based on two types which were forward SILAC and reverse SILAC. In forward SILAC, the HL-60 cells were cultured in light medium and treated with 10µM of lovastatin for 24 hours and the cells cultured in the heavy medium was used as an untreated control. In the reverse SILAC experiment, the cells were cultured in the heavy medium and were treated with lovastatin and the cells cultured in the light medium was used as a treated …show more content…
The proteins were in-gel digested with trypsin overnight forming peptides. The peptides were extracted with 5% acetic acid in H2O and 5% acetic acid in CH3CN/ H2O at a 1:1 ratio, dried and stored at -200C for further analysis.
B) Protein Identification A high performance liquid chromatography separation was used in presence of homemade trapping column and separation column. Both the column were packed with ReproSil-Pur C18-AQ resin. The determined peptide mixture was loaded into the trapping column with a solvent mixture of 0.1% formic acid at a flow rate of 4.0µL/min. The separation of the peptides were then carried out at 120 minutes in 2-4% acetonitrile and 0.1% formic acid at a flow rate of 300nL/min. A LC-MS/MS analysis was performed on an LTQ-Orbitrap Velos mass spectrometer and operated in a positive-ion mode at a voltage of 1.8kV. The twenty most abundant ions in mass spectrometry at a threshold above 500 counts were determined for fragmentation by collision-induced dissociation at a 35% collision energy.
4) Data analysis and verification