Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches

Abstract

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

“…Furthermore, the subunits of these proteasomes were separated by 2D gel electrophoresis and the protein spots from proteasome purified from the brain and spleen were identified by mass spectrometry. All spots between 20 and 30 kDa were matched to proteasomal subunits, indicating the purity of the preparations, and altogether, 14 subunits were identified in chicken as already found in humans, mice, and rats (Claverol et al 2002;Froment et al 2005;Schmidt et al 2006). Actually, the number of excised spots exceeded that of the proteasome subunits by 4, indicating that some proteins might be post-translationally modified, e.g., by glycosylation, phosphorylation, acetylation, or deamination and therefore have different properties, e.g., a different isoelectric point.…”

Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities

“…By means of 2D PAGE of 20S proteasomes, we had previously shown that liver contains proteasomes exclusively of an intermediate-type subunit composition, i.e., they contain standard as well as immuno-subunits (Schmidt et al 2006). Applying the same technique, we analysed the subunit composition of the three proteasome subpopulations of young and aged rats, respectively, and assigned the proteasome subunits according to our earlier investigations ( Fig.…”

“…Applying the same technique, we analysed the subunit composition of the three proteasome subpopulations of young and aged rats, respectively, and assigned the proteasome subunits according to our earlier investigations ( Fig. 3a) (Schmidt et al 2006). Relative quantification of the active site containing subunits β1 and β1i as well as of β5 and β5i was performed by using subunits α2 and β4 as reference spots.…”

“…The resulting supernatant (liver extract) was used for determination of protein and 20S proteasome content, for measurement of chymotrypsin-, trypsin-and caspase-like activities as already described as well as for purification of 20S proteasome as described elsewhere (Schmidt et al 2006). Briefly, this consists of the following steps: a fractionated protein precipitation with ammonium sulphate, chromatography on DEAE-Sephacel, gel filtration on Superose 6 and anion exchange chromatography on Mono Q.…”

“…An incomplete replacement of standard-subunits by immuno-subunits results in the formation of a third population designated intermediate-type proteasomes, which can be further subdivided into subpopulations according to the extent of standard by immuno-subunit replacement (Klare et al 2007;Dahlmann et al 2000;Guillaume et al 2010). We have shown previously that proteasomes of rat liver all comprised intermediate-type subunit compositions (Dahlmann et al 2000;Schmidt et al 2006). Taking advantage of their different surface hydrophobicity, we separated 20S proteasomes from rat heart into three subpopulations two of them comprising intermediate-type and one containing standard proteasomes (Kloss et al 2009).…”