Identification of the molecular composition of the 20S proteasome of mouse intestine by high-resolution mass spectrometric proteome analysis

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2009
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Preywisch, Regina
Youhnovski, Nikolay
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Methods in Molecular Biology. 2009, 564, pp. 173-186. ISSN 1064-3745. Available under: doi: 10.1007/978-1-60761-157-8_10
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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 α1 α7 and β1 β7 were completely and unambiguously identified. In addition to subunits β1 and β2, the corresponding inducible subunits being part of the immuno-proteasome were identified. The subunit β5i 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.

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570 Biowissenschaften, Biologie
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20S-Proteasome, High-resolution FTICR mass spectrometry, Proteome analysis, Proteasome constituents, UV- and IR-MALDI ionisation
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ISO 690WEBER, Reinhold, Regina PREYWISCH, Nikolay YOUHNOVSKI, Marcus GRÖTTRUP, Michael PRZYBYLSKI, 2009. Identification of the molecular composition of the 20S proteasome of mouse intestine by high-resolution mass spectrometric proteome analysis. In: Methods in Molecular Biology. 2009, 564, pp. 173-186. ISSN 1064-3745. Available under: doi: 10.1007/978-1-60761-157-8_10
BibTex
@article{Weber2009Ident-10054,
  year={2009},
  doi={10.1007/978-1-60761-157-8_10},
  title={Identification of the molecular composition of the 20S proteasome of mouse intestine by high-resolution mass spectrometric proteome analysis},
  volume={564},
  issn={1064-3745},
  journal={Methods in Molecular Biology},
  pages={173--186},
  author={Weber, Reinhold and Preywisch, Regina and Youhnovski, Nikolay and Gröttrup, Marcus and Przybylski, Michael}
}
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