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. The large-scale analysis of proteins was made possible by high-resolution protein and peptide separation technologies such as two-dimensional gel electrophoresis (2-DE) 1 (1) and capillary chromatography (2) combined with the development of matrix-assisted laser desorption ionization (MALDI) (3) and electrospray ionization (ESI) (4) mass spectrometry (MS), two soft ionization techniques that enable the analysis of large biomolecules. 2-DE allows the highest resolution of protein separation, with up to 10,000 spots (5). Typically, 2-DE was combined with protein identification via peptide mass fingerprinting using MALDI-MS, often supported by tandem MS (MS/MS) produced with post-source decay (6). In 1996, the term "proteome" was defined as the protein composition of a cell, organism, organelle, tissue, or body fluid at a given time (7). However, the proteome is not the direct complement of the genome because of alternative splice variants, post-trans- 1 The abbreviations used are: 2-DE, two-dimensional gel electrophoresis; emPAI, exponentially modified protein abundance index; ESI, electrospray ionization; H/L, heavy-to-light; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionizat...

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

“…Combined with enrichment techniques, 2-DE-MS and shotgun-MS may complement each other to characterize diverse forms of oxidation modifications [78]. However, multiple proteins can co-migrate in 2-DE, which means absolute quantification may not be possible [79]. In addition, hydrophobic proteins or proteins with high isoelectric point may be poorly resolved by 2-DE, although many of the earlier perceived limitations of 2-DE have now been either effectively addressed or established to be little more than dogma associated with poor sample handling and methodological practices.…”

Reconsidering Old Data: Non-canonical HspB1 Species and the Enigma of the Cytoskeletal Function of HspB1

“…To complicate things, a recent publication by Thiede et al ( 2013 ) demonstrated that ~50 % of 2-DE protein spots contained more than one identifi ed protein or protein species, using the most sensitive analytical methods available at present that included stable isotopic labeling with amino acids in cell culture (SILAC) and 2-DE-Orbitrap-LC mass spectrometry. This approach allowed the quantifi cation of proteins at the protein species level.…”

“…The above outlined isotopic labeling/2-DE-Orbitrap-LC mass spectrometry method provided evidence for the existence of multiple HspB1 species, and, most importantly, also for their specifi c regulation during apoptosis in HeLa cells (Thiede et al 2013 ). By this approach ~1,200 proteins were identifi ed, together with ~2,700 protein species.…”

“…Subsequently, the cell proteins were separated by 2-DE in a pH gradient from 2 to 11 and an Mr range from 3 to 150 kDa with 23 × 30 cm large gels. After staining of the 2-DE gels with CBB-G250, all visible spots were excised and analyzed by LC-Orbitrap mass spectrometry (Thiede et al 2013 ). The sector of the gel containing 13 spots of HspB1 species is shown; some of them were down-regulated ( red numbers ) by a factor (non-labeled/labeled) < 0.5, whereas others were up-regulated ( blue numbers ) by a factor > 2 during by apoptosis.…”

Towards the Full Realization of 2DE Power

“…It also seems that this scheme worked well in the case of uncomplicated proteomes, such as mycoplasma, for instance, where the expected numbers of proteoforms in proteomes were not so great as in mammalian cells [83,84,85,86]. After application of more sophisticated MS instruments for identification of proteins in 2DE spots (especially ESI LC-MS/MS), it was revealed that, depending on the gel resolution, the spots often contained more than a single protein, especially in the case of mammalian cells [87,88]. So, the quantitation of proteins became an ambiguous task.…”