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Title (Primary) 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
Author Schmidt, F.; Hustoft, H.K.; Strozynski, M.; Dimmler, C.; Rudel, T.; Thiede, B.;
Journal Electrophoresis
Year 2007
Department PROTEOM; MOLSYB;
Volume 28
Issue 23
Language englisch;
Keywords apoptosis; cisplatin; LC-MALDI; p54nrb; stable isotope labeling with amino acids in cell culture
Abstract Quantitative proteome analysis of cisplatin-induced apoptosis in total JurkatTcell 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 off line 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)down arrow D, DQVD(286)down arrow R, and MMPD422 down 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)down arrow D) within the NOPS domain of p54nrb. Concisely, quantitative protein data generated by off line 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
ID 2267
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=2267
Schmidt, F., Hustoft, H.K., Strozynski, M., Dimmler, C., Rudel, T., Thiede, B. (2007):
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
Electrophoresis 28 (23), 4359 - 4368