Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells.

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin-proteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC-ESI-MS. Taking the results of all experiments together, no quantitative changes were observed for the α- and ß-subunits of the 20S proteasome except for subunit α7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (α7a) and up-regulation of the more basic protein species (α7b) during apoptosis. The difference in these two α7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in α7a, whereas these modifications were missing in α7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit α7 after induction of apoptosis.

Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI-MS.

A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI-MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5.

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.

Quantitative proteome analysis of cisplatin-induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS-PAGE, and LC-MALDI-TOF/TOF MS.

Quantitative proteome analysis of cisplatin-induced apoptosis in total Jurkat T cell lysates was performed in order to identify modified proteins. Proteins were labeled in cell culture with stable isotopes of arginines, and fractionated by SDS-PAGE. Subsequently, tryptic peptides were analyzed by nano-LC coupled offline to MALDI-TOF/TOF-MS as an alternative to commonly used online LC-ESI-MS. As a result, 26 proteins were found with a relative abundance higher than 1.5, thereof 19 already known and seven unknown to be involved in apoptosis (adenine phosphoribosyltransferase, microsomal signal peptidase 25 kDa subunit, phosphomevalonate kinase, probable rRNA processing protein EBP2, RNA-binding protein 4, transmembrane protein 33, and tetratricopeptide repeat domain 9C). Immunoblotting of core-binding factor beta and elongation factor 2 revealed similar quantitative changes as detected by the SILAC-based proteomics approach. Strikingly, 8 of 26 identified apoptosis-modified proteins contained at least one RNA-binding motif. Three caspase cleavage sites of the 54 kDa nuclear RNA-binding protein (p54nrb) were mapped at DQLD(231) (downward arrow)D, DQVD(286) (downward arrow)R, and MMPD(422) (downward arrow)G by applying caspase-3 to the in vitro translated protein and mutation analysis. The determined caspase cleavage sites were located C-terminal to the two RNA-binding motifs and one (DQLD(231) (downward arrow)D) within the NOPS domain of p54nrb. Concisely, quantitative protein data generated by offline LC-MALDI-MS were shown to be particularly accurate. Furthermore, only regulated peptides were selected in a result-dependent manner for MS/MS analyses and revealed novel apoptosis-modified proteins.