Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches.
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/.
Schmidt
Frank
F
Max Planck Institute for Infection Biology, Core Facility Protein Analysis, Berlin, Germany.
Dahlmann
Burkhardt
B
Janek
Katharina
K
Kloss
Alexander
A
Wacker
Maik
M
Ackermann
Renate
R
Thiede
Bernd
B
Jungblut
Peter R
PR
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D007553:Isotope Labeling; D008099:Liver / Q000737:chemistry*; D008110:Liver Extracts / Q000737:chemistry; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000032:analysis*; D021122:Protein Subunits / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008110:Liver Extracts; D021122:Protein Subunits; D020543:Proteome; D046988:Proteasome Endopeptidase Complex