ORIGINAL ARTICLE Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells Frank Schmidt • Burkhardt Dahlmann • Hanne K. Hustoft • Christian J. Koehler • Margarita Strozynski • Alexander Kloß • Ursula Zimny-Arndt • Peter R. Jungblut • Bernd Thiede Received: 1 December 2009 / Accepted: 17 March 2010 / Published online: 3 April 2010 Ó Springer-Verlag 2010 Abstract 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 exe- cuted 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 a- and b-subunits of the 20S proteasome except for subunit a7. This protein was iden- tified in two protein spots with a down-regulation of the more acidic protein species (a7a) and up-regulation of the more basic protein species (a7b) during apoptosis. The difference in these two a7 protein species could be attrib- uted to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in a7a, whereas these modifications were missing in a7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit a7 after induction of apoptosis. Keywords Apoptosis Á 5-Fluorouracil Á 20S proteasome Á Phosphorylation Á SILAC Abbreviations 19S-Reg 19S regulator complex 5-FU 5-Fluorouracil H/L Heavy to light PARP1 Poly (ADP-ribose) polymerase-1 SILAC Stable isotope labeling with amino acids in cell culture Introduction Many important intracellular processes are regulated by transcription, translation and protein degradation. In eukaryotic cells, the ubiquitin–proteasome pathway is the major non-lysosomal pathway for protein degradation. This proteolytic pathway is based on the coordinated and syn- ergistic activity of two enzymatic systems: proteins are initially targeted for proteolysis by the attachment of a polyubiquitin chain catalyzed by the ubiquitin-conjugating system and are then recognized and rapidly degraded to small peptides by the proteasome (Hershko and This article is published as part of the Special Issue on Protein Species and Time Schedule. Electronic supplementary material The online version of this article (doi:10.1007/s00726-010-0575-6) contains supplementary material, which is available to authorized users. F. Schmidt Á H. K. Hustoft Á C. J. Koehler Á M. Strozynski Á B. Thiede (&) The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, Blindern, P.O. Box 1125, 0317 Oslo, Norway e-mail: bernd.thiede@biotek.uio.no F. Schmidt Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany B. Dahlmann Á A. Kloß Institute of Biochemistry, Charite ´-Universita ¨tsmedizin, Berlin, Germany U. Zimny-Arndt Á P. R. Jungblut Max Planck Institute for Infection Biology, Core Facility Protein Analysis, Berlin, Germany 123 Amino Acids (2011) 41:351–361 DOI 10.1007/s00726-010-0575-6