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Submitted on September 14, 2005
Revised on October 27, 2005
Accepted on November 10, 2005

An integrated mass spectrometry-based proteomics approach -QTAX to decipher the 26S proteasome interacting network

Cortnie Guerrero, Christian Tagwerker, Peter Kaiser, and Lan Huang

Department of Physiology & Biophysics, University of California, Irvine, Irvine, CA 92697-4560

Corresponding Author: lanhuang{at}uci.edu

We have developed an integrated proteomics approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26S proteasome interaction network in yeast. We have termed this approach QTAX for quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes. For this work, in vivo formaldehyde cross-linking was employed to freeze both stable and transient interactions occurring in intact cells prior to lysis. To isolate cross-linked protein complexes with high purification efficiency under fully denaturing conditions, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification. Tandem-affinity purification after in vivo cross-linking was combined with tandem mass spectrometry coupled with a quantitative SILAC strategy to carry out unambiguous protein identification and quantification of specific protein interactions. Using this method, we have captured and identified the full composition of yeast 26S proteasome complex as well as the two known ubiquitin receptors, Rad23 and Dsk2. Quantitative mass spectrometry analysis allowed us to distinguish specific proteasome interacting proteins (PIPs) from background proteins and led to the identification of a total of 64 potential PIPs, of which 42 are novel interactions. Among the 64 putative specific PIPs, there are ubiquitin pathway components, ubiquitinated substrates, chaperones, and transcription and translation regulators, demonstrating the efficacy of the developed approach in capturing in vivo protein interactions. The method offers an advanced technical approach to elucidate the proteasome’s dynamic protein interaction networks, and can find a wide range of applications in the studies of other macromolecular protein complex interaction networks.


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