An Integrated Mass Spectrometry‐Based Proteomics Approach‐QTAX to Decipher the 26S Proteasome Interacting Network

Abstract

We have developed an integrated proteomics approach-QTAX to decipher in vivo protein-protein interactions in the cellular environment and applied this strategy to globally map the 26S proteasome interaction network. We employed in vivo formaldehyde cross-linking to freeze both stable and transient interactions occurring in intact cells prior to lysis. To achieve high purification efficiency under fully denaturing condition, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification of cross-linked products. 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. With this method, we have captured and identified the full composition of yeast 26S proteasome complex as well as the two known soluble ubiquitin receptors. 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 43 are novel 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 interaction networks of other macromolecular protein complexes.