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...

Since its introduction, isobaric peptide labeling has played an important role in relative quantitative comparisons of proteomes. This paper describes isobaric peptide termini labeling (IPTL), a novel approach for the identification and quantification of two differentially labeled states using MS/MS spectra. After endoproteinase Lys-C digestion, peptides were labeled at C-terminal lysine residues with either 2-methoxy-4,5-dihydro-1H-imidazole (MDHI) or with tetradeuterated MDHI-d(4). Subsequently, their N-termini were derivatized either with tetradeuterated succinic anhydride (SA-d(4)) or with SA. The mixed isotopic labeling results in isobaric masses and provided several quantification data points per peptide. The suitability of this approach is demonstrated with MS and MS/MS analyses of Lys-C digests of standard proteins. A conceptually simple quantification strategy with a dynamic range of 25 is achieved through the use of Mascot score ratios. The utility of IPTL for the analysis of proteomes was verified by comparing the well-characterized effect of the antimitotic inhibitor S-Trityl-l-Cysteine (STLC) on HeLa cells that were treated for either 24 or 48 h with the inhibitor. Many apoptosis-linked proteins were identified as being differentially regulated, confirming the suitability of IPTL for the analysis of complex proteomes.

Recently, we introduced a novel approach for protein quantification based on isobaric peptide termini labeling (IPTL). In IPTL, both peptide termini are dervatized in two separate chemical reactions with complementary isotopically labeled reagents to generate isobaric peptide pairs. Here, we describe a novel procedure for the two chemical reactions to enable a cost-effective and rapid method. We established a selective N-terminal peptide modification reaction using succinic anhydride. Dimethylation was used as second chemical reaction to derivatize lysine residues. Both reactions can be performed within 15 min in one pot, and micropurification of the peptides between the two reactions was not necessary. For data analysis, we developed the force-find algorithm in IsobariQ which searches for corresponding peaks to build up peak pairs in tandem mass spectrometry (MS/MS) spectra where Mascot could not identify opposite sequences. Utilizing force-find, the number of quantified proteins was improved by more than 50% in comparison to the standard data analysis in IsobariQ. This was applied to compare the proteome of HeLa cells incubated with S-trityl-L-cysteine (STLC) to induce mitotic arrest and apoptosis. More than 50 proteins were found to be quantitatively changed, and most of them were previously reported in other proteome analyses of apoptotic cells. Furthermore, we showed that the two complementary isotopic labels coelute during liquid chromatography (LC) separation and that the linearity of relative IPTL quantification is not affected by a complex protein background. Combining the optimized reactions for IPTL with the open source data analysis software IsobariQ including force-find, we present a straightforward and rapid approach for quantitative proteomics.

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.