The majority of proteins exert an effect in the context of macromolecular assemblies that are part of dynamic networks of enormous complexity. Cellular processes, such as cell signalling, proliferation, apoptosis and cell growth, emerge to a large extent from the properties of such networks. Hence, understanding and modelling of cellular processes in healthy and pathological conditions depend on comprehensive and robust information on the topology and the dynamic properties of the engaged protein networks. Initially, large-scale protein interaction studies were performed with the yeast two-hybrid technology, which provided insights into global patterns of binary protein interactions of model organism proteomes (Uetz et al, 2000
; Walhout et al, 2000
; Ito et al, 2001
). More recently, affinity purification coupled with mass spectrometry (AP-MS) has become the method of choice for the analysis of protein complexes under near-physiological conditions (Gingras et al, 2007
; Kocher and Superti-Furga, 2007
). Large-scale AP-MS studies performed in yeast provided the first comprehensive set of high-density interaction data, which became an invaluable source of information for yeast systems biology (Gavin et al, 2002
; Ho et al, 2002
; Krogan et al, 2006
). The success in yeast can be mainly attributed to the high efficiency of homologous recombination that allowed genome-wide tagging of yeast ORFs as a valuable resource for large-scale AP-MS studies. However, no such genetic system exists for multicellular eukaryotes.
Given the various cell types and cellular states, each characterized by specific protein–protein interaction networks, the complexity of the human proteome and the limited genetic methods available to generate cell lines expressing affinity-tagged proteins, global analysis of protein complexes and protein interaction networks in human cells is a daunting task. Progress towards this goal will strongly depend on efficient and robust AP-MS workflows for human cells that provide comprehensive as well as high-confidence protein complex information to populate public databases. The robustness and reproducibility of such methods are key because it can be anticipated that data from different studies and research groups must be combined to achieve saturation coverage of the human interaction proteome. However, false discovery and reproducibility rates are not known for existing methods, which make the combination of AP-MS data from different studies difficult. In addition, present AP-MS strategies are limited by the labour-intense generation of large collections of human cell lines for expression of epitope-tagged bait proteins, the low yield of protein complex isolation from such cell lines and the limited sensitivity of MS-based protein identification.
To overcome some of these major limitations, we have developed an integrated experimental workflow. Besides optimizing each experimental step, we focused on the compatibility of the steps with each other to generate a process with improved performance. As a result, the proposed procedure significantly enhanced the throughput of generating bait-expressing cell lines, increased the protein complex purification yields by a novel double-affinity strategy and allowed analysis of protein complexes and interaction networks with high sensitivity and reproducibility. We applied this procedure to study a network of human protein phosphatase 2A (PP2A) complexes. PP2A is a heterotrimeric, evolutionary conserved serine/threonine phosphatase with regulatory functions in a wide range of cellular processes, including transcription, apoptosis, cell growth and cellular transformation (Virshup, 2000
; Lechward et al, 2001
). The human genome encodes two catalytic subunits (PPP2CA, PPP2CB), two scaffolding subunits (PPP2R1A, PPP2R1B) and at least 15 known regulatory B subunits that, by combinatorial assembly, can potentially form a multitude of different trimeric PP2A complexes (Janssens and Goris, 2001
; Lechward et al, 2001
). It is believed that the versatile nature of this combinatorial subunit arrangement provides substrate specificity as well as temporal and spatial control of phosphatase activity. However, no systematic study has yet been performed to address the question, which PP2A complex forms indeed coexist in human cells and how these complexes are connected to specific cellular processes through protein–protein interactions. Using the method described in this work, we identified 197 specific protein–protein interactions at a reproducibility rate of at least 85%. The discovered interactions constitute a network of different classes of concurrently present phosphatase complexes that in turn are linked to proteins with specific functions in cell signalling, mitosis, DNA repair and more.
On the basis of these results, we believe that the proposed analytical procedure will significantly improve the scope and reproducibility of future AP-MS studies on the human interaction proteome.