Identification of the molecular composition of the 20S proteasome of mouse intestine by high-resolution mass spectrometric proteome analysis.
In the last years, intracellular protein degradation by the proteasome has become a focus area of scientific interest. Here, we describe a proteomics approach for the molecular mapping of the constituents of the proteolytically active core particle, the constitutive 20S proteasome from mouse intestine. In addition to the proteomics workflow widely used for protein isolation, gel electrophoretic separation, in-gel digestion, and UV-MALDI mass spectrometry, high-resolution Fourier transform ion cyclotron resonance mass spectrometry using infrared-MALDI ionisation (IR-MALDI FTICR-MS) has been employed as an efficient method for protein identification by peptide mass fingerprint. The 20S proteasome subunits alpha1-alpha7 and beta1-beta7 were completely and unambiguously identified. In addition to subunits beta1 and beta2, the corresponding inducible subunits being part of the immuno-proteasome were identified. The subunit beta5i was found to completely replace the corresponding constitutive subunit, suggesting a high proteolytic activity of the intestinal proteasome leading to increased production of antigenic peptides. The high mass accuracy in the low ppm range and resolution of FTICR-MS provide direct identifications of individual proteins as mixtures such as components resulting from incomplete electrophoretic separation. In addition, the comparison of UV- and IR-MALDI FTICR-MS may provide details of fragmentation and rearrangement reactions that may occur under UV-MALDI ionisation conditions.
Weber
Reinhold
R
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, Konstanz, Germany.
Preywisch
Regina
R
Youhnovski
Nikolay
N
Groettrup
Marcus
M
Przybylski
Michael
M
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D007422:Intestines / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D046988:Proteasome Endopeptidase Complex / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D010446:Peptide Fragments; D020543:Proteome; D046988:Proteasome Endopeptidase Complex