The team, led by Harvard's Steven Gygi turned to a high-throughput affinity-purification mass spectrometry approach to uncover interacting protein partners for more than 2,500 proteins in a human cell line. From this, they developed a network, dubbed BioPlex, that includes 23,744 interactions among 7,668 proteins. Some 86 percent of these interactions had not been reported previously.
This, they noted, is the first phase of an effort to profile the entire human ORFEOME through AP-MS to generate a comprehensive map of protein interactions in humans.
"Ultimately, BioPlex unveils both individual protein functions and global proteome organization," Gygi and his colleagues wrote in their paper.
Mapping the human interactome poses a number of challenges, but Gygi and his colleagues argued that an AP-MS approach would give a good "first-pass" glimpse of it as the method is fairly sensitive and can determine components of multi-protein complexes.
For this project, they employed high-throughput lentiviral expression and AP-MS profiling of C-terminal FLAG-HA-tagged baits representing 13,000 protein-coding ORFs from the Human ORFEOME. Each month, they expressed up to 600 baits in HEK293T cells that were then immune-purified and analyzed with a mass spec, they reported.
The analysis of all the protein-coding ORFs is ongoing, Gygi and his colleagues said, noting that this analysis focuses on the first 2,594 AP-MS experiments.
With an updated version of the comparative proteomics analysis software CompPASS, they sorted out interacting pairs of proteins from the background.
Though each AP-MS experiment yields data on associations with one bait, when combined, the experiments sketch out a model of the interactome.
Here, the researchers reported on 23,744 interactions that connect 7,688 gene products, a network they dubbed the BioPlex.
When the researchers overlaid their BioPlex network onto mammalian protein complexes in the CORUM database, they found that more than a quarter of CORUM complexes were perfectly recapitulated by BioPlex while about half had 80 percent coverage, indicating that it has correctly mapped the interactome.
Additionally, a number of the interactions in BioPlex hadn't been previously reported. In a bid to validate those, the researchers confirmed that the protein pairs actually were localized to the same subcellular region — making it plausible that they can interact — and then calculated the assortativities of several databases and interaction datasets and compared them to BioPlex, finding that it compared favorably and exhibited a greater tendency to connect co-localized proteins.
Gygi and his colleagues also mapped the BioPlex community structure using clique percolation to find 256 communities that could then be further divided into 354 communities and sub-communities. These communities typically were enriched for one gene ontology term or Pfam domain.
For instance, after clique perforation, the proteasome was represented by one large community, but further modularity-based clustering grouped it into two parts that largely corresponded to its catalytic and regulatory particles and to its ubiquitin ligase complex.
Other communities matched known complexes, the researchers added.
As the researchers noted earlier, proteins that interact have to be found near one another. Thus, they added, the interactome reflects the subcellular localization of the proteins.
After mapping subcellular localization information from UniProt onto BioPlex, the researchers calculated the enrichment of subcellular compartments among each protein's neighbors. If its partners are all in one region, then that protein likely spends some time there, too, providing some insight into its biology.
Proteasome subunits, for example, were pinpointed to both nuclear and cytoplasmic regions.
To apply BioPlex to a clinical issue, the researchers examined the sub-network that includes VAPB, which is mutated in some familial ALS patients. VAPB and several of its interacting partners, the researchers said, were associated with oxysterol-binding proteins and other proteins associated with membrane traffic or signaling.
To validate these interactions, the researchers expressed VAPB and its mutant forms in a cell line and then performed AP-MS. From this and other experiments, they noted that the mutant form of the protein has increased interactions with some proteins like LSG1 but has decreased interactions with proteins with FFAT motifs.
"These data demonstrate the potential for BioPlex to inform and enhance focused study of individual proteins," the researchers said.
Gygi and his colleagues noted, though, that as BioPlex is based on a single cell type, protein interactions could vary in other lineages.
"Thus, the network is best viewed as a framework that can be used for hypothesis generation and for design and interpretation of targeted studies that address dynamic and genetic underpinnings of individual networks, as illustrated through our quantitative analysis of the VAPB complex," they added.