High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer

Abstract

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography 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, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the 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 stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows.

Fig. 1.

The SILAC/2-DE/nano-LC-MS workflow.

Fig. 2.

Overview of relation of unique proteins, protein spots, and protein abundance. Diagrams of the distribution of the number of identified proteins per spot (A), the number of spots where a unique protein was identified (B), and the exponentially modified protein abundance index (emPAI) values (%) of the identified proteins within a 2-DE spot are displayed.

Fig. 3.

2-DE spot pattern of lamin A/C. A, protein spots in which lamin A/C was identified and quantified are indicated by spot numbers. B, the identified peptides within each spot covering the sequence of lamin A are presented in yellow, and if both the ¹²C₆ (light, apoptosis) and the ¹³C₆ (heavy, control) forms of arginine and lysine were identified, they are shown in green. Spot numbers in which lamin A/C was quantified are shown with a fold change H/L of <0.188 in bold red, 0.188–0.5 in red, 0.5–2 in black, 2–3.795 in blue, and >3.796 in bold blue. Putative caspase cleavage sites are shown at the bottom. C, immunoblots of control and STLC-treated HeLa cells using antibodies against lamin A/C, lamin A/C (pSer392), and lamin A/C (pSer22).

Fig. 4.

2-DE spot pattern of vimentin. A, protein spots in which vimentin could be identified and quantified are indicated by spot numbers. B, the identified peptides within each spot covering the sequence of vimentin are presented in yellow, and if both the ¹²C₆ (light, apoptosis) and the ¹³C₆ (heavy, control) forms of arginine and lysine were identified, they are shown in green. Spot numbers in which vimentin was quantified are shown with a fold change H/L of <0.188 in bold red, 0.188–0.5 in red, 0.5–2 in black, 2–3.795 in blue, and >3.796 in bold blue. Putative caspase cleavage sites are shown at the bottom.