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Analysis of the Plasmodium falciparum proteasome using Blue Native PAGE and label-free quantitative mass spectrometry.

Amino acids (2012-07-24)
Nicole Sessler, Karsten Krug, Alfred Nordheim, Benjamin Mordmüller, Boris Macek
ABSTRACT

Detailed knowledge of the composition of protein complexes is crucial for the understanding of their structure and function; however, appropriate techniques for compositional analyses of complexes largely rely on elaborate tagging, immunoprecipitation, cross-linking and purification strategies. The proteasome is a prototypical protein complex and therefore an excellent model to assess new methods for protein complex characterisation. Here we evaluated the applicability of Blue Native (BN) PAGE in combination with label-free protein quantification and protein correlation profiling (PCP) for the investigation of proteasome complexes directly from biological samples. Using the purified human 20S proteasome we showed that the approach can accurately detect members of a complex by clustering their gel migration profiles. We applied the approach to address proteasome composition in the schizont stage of the malaria parasite Plasmodium falciparum. The analysis, performed in the background of the whole protein extract, revealed that all subunits comigrated and formed a tight cluster with a single maximum, demonstrating presence of a single form of the 20S proteasome. This study shows that BN PAGE in combination with label-free quantification and PCP is applicable to the analysis of multiprotein complexes directly from complex protein mixtures.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Brilliant Blue G solution, Concentrate
Sigma-Aldrich
Brilliant Blue G, pure
Sigma-Aldrich
Brilliant Blue G, 250, for microscopy