An integrated mass spectrometry-based proteomic approach -: Quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network

We developed an integrated proteomic approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26 S proteasome interaction network in yeast. We 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 used 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 hexa-histidine 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 (stable isotope labeling of amino acids in cell culture) strategy to carry out unambiguous protein identification and quantification of specific protein interactions. Using this method, we captured and identified the full composition of yeast 26 S 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 dynamic protein interaction networks of the proteasome and can find a wide range of applications in the studies of other macromolecular protein complex interaction networks.