Protein species high resolution quantitative proteomics of HeLa cells using SILAC-2-DE-nanoLC/LTQ-Orbitrap mass spectrometry
The proteomics field has shifted during the last years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel free workflows due to the tremendous developments in isotopic labelling techniques, nano-liquid chromatography (LC) and high resolution mass spectrometry (MS). However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined SILAC of controls and apoptotic HeLa cells with 2-DE and subsequent analysis of tryptic peptides by nano-LC coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins, however, up to 23 proteins were identified in a single 2-DE spot. Only half of the 2-DE spots represented a dominant protein with more than 90% of whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise and minor components could not be quantified. These problems are circumvented by quantification using SILAC. Despite challenging as shown in detail for lamin A/C and vimentin, quantitative changes of protein species can be detected. Combining 2-DE with high resolution LC-MS identified proteomic changes in apoptotic cells that are unobservable using any of the other previously employed proteomic workflows.