Abstract:
The disclosure relates to a virus-like particle in which a protein complex is entrapped, ensuring the formation of the protein complex under physiological conditions, while protecting the protein complex during purification and identification. The disclosure further relates to the use of such virus-like particle for the isolation and identification of protein complexes.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2013/060787, filed May 24, 2013, designating the United States of America and published in English as International Patent Publication WO 2013/174999 A1 on Nov. 28, 2013, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 12169209.9, filed May 24, 2012. 
     
    
     TECHNICAL FIELD 
       [0002]    The disclosure relates generally to biotechnology, and more particularly to a virus-like particle in which a protein complex is entrapped, ensuring the formation of the protein complex under physiological conditions, while protecting the protein complex during purification and identification. The disclosure further relates to the use of such virus-like particle for the isolation and identification of protein complexes. 
       BACKGROUND 
       [0003]    Protein-protein interactions are an essential key in all biological processes, from the replication and expression of genes, to the morphogenesis of organisms. Protein-protein interactions govern, amongst others, ligand-receptor interaction and the subsequent signaling pathway; they are important in assembly of enzyme subunits, in the formation of biological supramolecular structures such as ribosomes, filaments and virus particles, and in antigen-antibody interactions. 
         [0004]    Researchers have developed several approaches in attempts to identify protein-protein interactions. A major breakthrough was obtained by the introduction of the genetic approaches, of which the yeast two-hybrid (Fields and Song, 1989) is the most important one. Although this technique became widely used, it has several drawbacks. The fusion proteins need to be translocated to the nucleus, which is not always evident. Proteins with intrinsic transcription activation properties may cause false positives. Moreover, interactions that are dependent upon secondary modifications of the protein such as phosphorylation cannot be easily detected. 
         [0005]    Several alternative systems have been developed to solve one or more of these problems. 
         [0006]    Approaches based on phage display do avoid nuclear translocation. WO9002809 describes how a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, wherein the gene encoding the binding protein is packaged inside the phage. Phages that bear the binding protein that recognizes the target molecule are isolated and amplified. Several improvements of the phage display approach have been proposed, as described, e.g., in WO9220791, WO9710330, and WO9732017. 
         [0007]    However, all these methods suffer from the difficulties that are inherent at the phage display methodology: the proteins need to be exposed at the phage surface and are so exposed to an environment that is not physiologically relevant for the in vivo interaction. Moreover, when screening a phage library, there will be a competition between the phages that results in a selection of the high-affinity binders. 
         [0008]    U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybrid system, whereby it can be screened for modification-dependent protein-protein interactions. However, this method relies on the co-expression of the modifying enzyme, which will exert its activity in the cytoplasm and may modify enzymes other than the one involved in the protein-protein interaction, which may, on its turn, affect the viability of the host organism. 
         [0009]    An interesting evolution is described in U.S. Pat. No. 5,776,689, by the so-called protein recruitment system. Protein-protein interactions are detected by recruitment of a guanine nucleotide exchange factor (Sos) to the plasma membrane, where Sos activates a Ras reporter molecule. This results in the survival of the cell that otherwise would not survive in the culture conditions used. Although this method has certainly the advantage that the protein-protein interaction takes place under physiological conditions in the submembrane space, it has several drawbacks. Modification-dependent interactions cannot be detected. Moreover, the method is using the pleiotropic Ras pathway, which may cause technical complications, such as the occurrence of false positives. 
         [0010]    A major improvement in the detection of protein-protein interactions was disclosed in WO0190188, describing the so-called Mappit system. The method, based on a cytokine receptor, not only allows a reliable detection of protein-protein interactions in mammalian cells, but also modification-dependent protein interactions can be detected, as well as complex three-hybrid protein-protein interactions mediated by a small compound (Caligiuri et al., 2006). However, although very useful, the system is limited in sensitivity and some weak interactions cannot be detected. Moreover, as this is a membrane-based system, nuclear interactions are normally not detected. Recently, a cytoplasmic interaction trap has been described, solving several of those shortcomings. However, all of these “genetic” systems rely on the overexpression of both interaction partners, which may result in false positives due to the artificial increase in concentration of one or both of the interaction partners. 
         [0011]    As an alternative for the genetic protein-protein interaction detection methods described above, biochemical or co-purification strategies, combined with mass spectrometry-based proteomics (Paul et al., 2011; Gingras et al., 2007), can be used. For the co-purification strategies, a cell homogenate is typically prepared by a detergent-based lysis protocol, followed by capture using a (dual) tag approach (Gavin et al., 2002) or via specific antibodies (Malovannaya et al.). The protein complex extracted from the “soup” of cell constituents is then expected to survive several washing steps, mostly to compensate for the sensitivity of contemporary MS instruments, before the actual analysis occurs. There are no clear guidelines on the extent of washing or on available buffers and their stringency. Most lysis and washing protocols are purely empirical in nature and were optimized using model interactions. It is, therefore, hard to speculate on the loss of factors during these steps (false negatives), or the possibility of false interactions due to loss of cellular integrity (false positives). Use of metabolic labeling strategies allows separation between the proteins sticking to the purification matrix, and between the proteins that associate specifically to the bait protein. Depending on the purification conditions and the sensitivity of the MS instruments used, it is no exception to find more than 1000 proteins in the eluted fraction of a gel-free AP-MS experiment. 
         [0012]    There is a further need for co-purification techniques, isolating the protein complexes in their physiological environment, but wherein the complex is protected during the further purification and analysis. 
         [0013]    The evolutionary stress on viruses promotes highly condensed coding of information and maximal functionality for small genomes. Accordingly, for HIV-1, it is sufficient to express a single viral protein, the p55 GAG protein, to allow the efficient production of virus-like particles (VLPs) from cells (Gheysen et al., 1989; Shioda and Shibuta, 1990). The p55 GAG protein consists of different parts, which are processed by HIV protease upon maturation of the particle into a functional infectious particle. The N-terminal matrix protein part ensures binding to the membrane via myristoylation and ensures budding (Bryant and Ratner, 1990). The Capsid protein forms the cone-shaped viral core after processing, while the nucleocapsid protein and the p6 protein bind to and protect the viral RNA. The p55 GAG protein is highly mobile before accumulation in cholesterol-rich regions of the membrane, where multimerization actually initiates the budding process (Gomez and Hope, 2006). A total of 4000-5000 GAG molecules are required to form a single particle with a size of about 145 nm (Briggs et al., 2004). 
       BRIEF SUMMARY 
       [0014]    Surprisingly, it was found that the p55 GAG protein can be used to trap a bait protein together with its physiological binding partners into VLPs that are budded from human cells. The very mild “extraction” or “abduction” of the protein complex ensures the identification of relevant interacting proteins. After introduction of a simple one-step particle enrichment protocol to speed up the workflow, it was found that this viral particle-based protein-protein interaction trap approach (called “Virotrap”) can be used for the detection of binary interactions. The identification of new partners by the coupling of the Virotrap process to MS-based analysis is also shown. 
         [0015]    A first aspect of the disclosure is an artificial virus-like particle (called “Virotrap particle”), comprising (1) a viral particle-forming polypeptide, (2) a first interaction polypeptide and (3) a second interaction polypeptide, interacting with the first interacting polypeptide. As explained below, in one preferred embodiment, the viral-forming polypeptide and the first interacting polypeptide may be two different polypeptide domains of a fusion protein (i.e., a fusion protein consisting of at least two polypeptides derived from two different proteins). 
         [0016]    In another preferred embodiment, the viral particle-forming polypeptide and the first interacting polypeptide are independent proteins. Besides the first and the second interaction polypeptide, the virus-like particle may comprise other proteins, recruited to first and/or second interaction polypeptides, wherein all the proteins together form one protein complex. “Polypeptide” refers to a polymer of amino acids and does not refer to a specific length of the molecule. This term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation. A “virus-like particle,” as used herein, is a particle consisting at least of a viral particle-forming protein, but preferably without the viral DNA or RNA. “Viral particle-forming proteins,” as used herein, are known to the person skilled in the art and are proteins that allow the assembly of viral particles and, preferably, budding of the particles of the cell. Examples of such particles have been described in the art and include, but are not limited to, particles derived from virus families including Parvoviridae (such as adeno-associated virus), Retroviridae (such as HIV), and Flaviviridae (such as Hepatitis C virus). 
         [0017]    In a preferred embodiment, the particle-forming polypeptide may be a modification of the naturally occurring particle-forming protein, such as a deletion and or mutation, as long as they do not inhibit the particle formation. Preferably, the deletion and/or mutation is reducing the binding of the particle-forming polypeptide with host proteins. Preferably, the modification is a fusion protein. Preferably, the viral particle-forming polypeptide, or its modification, forms a viral structure, consisting of a hollow particle, in which the first and second interaction polypeptides are trapped. In a preferred embodiment, the first interaction polypeptide is anchored to the viral structure, ensuring the capturing of the protein complex formed by the first and the second interaction polypeptide into the inside of the virus-like particle. The anchoring may be direct, wherein the fusion partner of the viral particle-forming polypeptide acts as the first interaction polypeptide, or indirect, wherein an independent linker molecule binds to the viral particle-forming polypeptide at one hand, and to a construct comprising the first interaction polypeptide at the other hand (“dimerizing linker”) as illustrated in  FIG. 1 . As a non-limiting example, such a linker may be a molecule as illustrated in  FIG. 1 , or it may be a bispecific protein or peptide affinity ligand such as an antibody, or in a preferred setting, a bispecific NANOBODY® or ALPHABODY® binding to the viral particle-forming polypeptide at one hand, and to the first interaction polypeptide at the other hand. In case of a direct anchoring, the viral-forming polypeptide and the first interacting polypeptide are two different polypeptide domains of the same protein. Preferably, the viral particle-forming polypeptide is a HIV protein; even more preferably, the viral particle-forming polypeptide is the p55 GAG protein, or a modification or functional fragment thereof. A “modification” or “functional fragment,” as used herein, is a modification or functional fragment that is still capable of forming virus-like particles that are capable of entrapping the protein complex according to the disclosure. Preferably, the modification is a fusion protein; even more preferably, the p55 GAG protein is fused to the first interaction polypeptide. 
         [0018]    Another aspect of the disclosure is the use of an artificial virus-like particle, according to the disclosure, for the detection of protein-protein interactions. 
         [0019]    Still another aspect of the disclosure is a method for detecting protein-protein interactions, the method comprising (1) the expression of a viral particle-forming polypeptide in a cell, (2) recruiting a first interaction polypeptide to the viral particle-forming polypeptide, (3) recruiting a second interacting polypeptide to the first interaction polypeptide, (4) isolation of the virus-like particles, and (5) analysis of the entrapped protein complex. Preferably, the cell is a mammalian cell. Preferably, the analysis of the entrapped protein complex is an MS-based analysis. It is clear for the person skilled in the art that protein-protein interactions of any nature can be detected with the method. As a non-limiting example, the method may be used to detect proteins involved in a signaling network, but it may also be used to detect antigen-antibody interactions, or other affinity-binding proteins and their target(s). 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0020]      FIG. 1 : Principle of conditional protein complex trapping in virotrap particles. A bait protein is fused in frame to FKBP12. Upon addition of Mtx-PEG-FK506 dimerizer, the FKBP 12-bait fusion protein is recruited to the GAG-eDHFR fusion protein, leading to trapping of the bait and its interacting proteins in the virotrap particles that are formed by GAG multimerization and budding. Further purification of virotrap particles followed by proteomic analysis results in identification of the interacting proteins. 
           [0021]      FIG. 2 : Plasmid map for the pMET7-VT1-MCS vector. Transfer of the coding sequences for EGFP led to the pMET7-VT1-EGFP vector, respectively, transfer of the GATEWAY® cassette resulted in the pMET7-VT1-GW, which then allowed transfer of the CSK1B coding sequence from a GATEWAY® entry clone, leading to pMET7-VT1-CKS1B. MCS: multi-cloning site 
           [0022]      FIG. 3 : Western blot showing the interaction between CSK1B bait and CDK2 prey upon addition of dimerizer (5 and 10 μM). HEK293T cells were transfected with the pMET7-VT1-CSK1B or pMET7-VT1-EGFP bait constructs, combined with the E-tagged CDK2 prey construct. After purification, the nanoparticles were directly brought in 2×SDS-PAGE loading buffer and loaded for SDS-PAGE separation and Western blot analysis. The three upper panels show samples prepared from purified particles. The lower panels are lysate samples obtained from the producer cells, confirming similar expression levels for the different components. 
           [0023]      FIG. 4 : Panel A: Schematic representation of the Virotrap strategy. Expression of a GAG-bait fusion protein results in the submembrane multimerization and consequent budding of virotrap particles from the cells. Interaction partners of the bait protein are also trapped in these virotrap particles and can be identified after purification and MS analysis. Panel B: Supernatants from HEK293T cells transfected with different combination of bait proteins (GAG-EGFP control and GAG-Ras) and FLAG-tagged prey proteins (IRS2 and RAF), were harvested after 24 hours, and were processed by ultracentrifugation to pellet the particles. Particle pellets were separated by SDS-PAGE and probed after Western blotting with anti-FLAG antibodies. Panel C: HEK293T cells were seeded in six-well plates and transfected with bait and prey combinations as in Panel B. Both wild-type and E-tagged VSV-G glycoproteins were expressed to allow particle enrichment via a single-step protocol from a 1 ml harvest. Western blotting of the eluted particles was performed with anti-FLAG and anti-GAG antibodies. Panel D: Virotrap experiments for two interaction pairs in both directions. Single-step purifications via anti-FLAG antibodies from six-well transfections were loaded for Western blotting and probed with anti-Etag, anti-GAG and anti-Actin antibodies for both the enriched particles and the producer cell lysates. The expression of GAG-S100A1 and GAG-S100B was below the detection limit in the lysates. Note that expression of the GAG protein without a fused bait does not lead to detectable particle formation. 
           [0024]      FIG. 5 : Detection of binary interactions with Virotrap. Comparison of the results for the positive (PRS) and the random reference set (RRS) obtained with the Virotrap system with other binary systems. All interactions from the PRS and RRS were explored by transfection in six-well plates, processing by a single-step purification protocol, and Western blot analysis of the eluted particles and lysates of the producer cells. The presence of prey proteins was revealed by anti-Etag antibodies on the VLP samples. The colored blocks show the Virotrap results for 30% positive interactions at the expense of 5% false positive signals in the RRS set. For the other methods, data from Braun et al., 2009, was used. 
           [0025]      FIG. 6 : Identification of novel interaction partners using co-complex MS analysis. Panel a: Analysis of the CDK2 interactome using Virotrap. A total of nine Virotrap experiments with on-bead VLP lysis was performed. The GAG-CDK2 identification list was challenged with identification lists from mock and GAG-EGFP controls, from GAG-FADD and from five additional Virotrap experiments. The CDK2 interactome (Right Panel Table) was obtained by removing all protein identifications found in the other experiments. *CKS1B was retained as it was also used as a GAG-BAIT construct in the reference experiments. Note that the CDK2-CKS1B interaction is a model interaction used for validation of the system. Panel b: The FADD interactome was obtained by adding three repeat experiments and controls using a specific elution protocol (scheme on the left). One of the FADD repeats was treated with TNFα during production. The protein list (right panel table) was obtained by considering only confident identifications (at least two peptides) and by removing all proteins identified in the eleven other experiments. The numbers in brackets show the number of times the protein was identified in the four FADD experiments. *: interaction was shown with Casein Kinase 1 α(CSNK1A). **Affects FAS signaling Panel c: Confirmation of the A20-FADD interaction. A20 was found in two FADD Virotrap experiments. The interaction was confirmed by co-immunoprecipitation experiments showing specific binding of A20 to immune-precipitated FADD (left panels), or of FADD to immune-precipitated A20 (right panels). Tagged proteins (FLAG and VSV tags) were expressed in HEK293T cells and were precipitated after lysis using paramagnetic anti-FLAG beads. The co-precipitated proteins were revealed by anti-VSV antibodies. 
           [0026]      FIG. 7 : Amino acid sequence of Desmoglein 1 (SEQ ID NO: 26) with annotation of extracellular, transmembrane and cytoplasmic regions, and with mapping of the peptides identified in the Virotrap analysis with S100A1 bait protein. 
       
    
    
     DETAILED DESCRIPTION 
     Examples 
     Materials and Methods 
     Plasmids and Antibodies 
       [0027]    The p55 GAG fusion constructs were generated by PCR amplification using primers Oligo1 and Oligo2 (see Table 1) of the p55 GAG coding sequence from the pCMV-dR8.74 packaging construct (Addgene) and by subsequent IN-FUSION® reaction (Clontech) in pMG1-Ras, a Ras expression vector used in the MAPPIT system (Eyckerman et al., 2001), resulting in a p55 GAG-RAS under control of the strong SRalpha promoter (pMET7-GAG-Ras). EGFP was transferred from pEGFP-C1 vector (Clontech) to generate the pMET7-GAG-EGFP construct. Using PCR-based cloning, a GATEWAY® cassette was inserted to allow recombination-assisted cloning. The complete set of positive and random reference clones were transferred in a single direction (no bait-prey swap) using standard GATEWAY® cloning. Prey ORFs from these sets were transferred into a GATEWAY®-compatible pMET7 expression vector with an N-terminal E-tag fused in frame. 
         [0028]    The pMD2.g pseudotyping vector was kindly provided by D. Trono. The pcDNA3-FLAG-VSV-G and pcDNA3-Etag-VSV-G are described elsewhere (Eyckerman et al., submitted). 
         [0029]    Antibodies used for Western blot were anti-p24 GAG (Abcam), anti-FLAG (M2, Sigma Aldrich), anti-actin (Sigma Aldrich) and anti-E-tag (Phadia). Secondary antibodies were from LI-COR, and blots were digitally imaged using an ODYSSEY® Imager system (LI-COR). 
       Cell Culture, Production and Purification of Virotrap Particles. 
       [0030]    HEK293T cells were cultured in a humidified atmosphere at 8% CO 2  using high-glucose DMEM (Invitrogen) complemented with 10% FCS and antibiotics. 
         [0031]    Cells were transfected overnight the day after seeding with a standard calcium phosphate transfection procedure. For ultracentrifugation experiments, 25 μg of bait vector (GAG-EGFP and GAG-Ras) was transfected and normalized to 50 μg with a mock vector, in 6×10 6  cells seeded the day before in 75 cm 2  bottles. For concentration of the virotrap particles, supernatant was harvested after 24 hours, centrifuged samples for 3 minutes at 1250×g to remove cellular debris and filtered the supernatant through 0.45 mm filters. The samples were then centrifuged in a Beckman ultracentrifuge using a Ti41 swinging bucket rotor at 22000 rpm. The supernatant was discarded and particle pellets were re-suspended directly in loading buffer for Western analysis. 
         [0032]    For binary interaction assays, 650,000 HEK293T cells were seeded the day before transfection in six-well plates. On the day of transfection, a DNA mixture was prepared containing the following: 3.5 μg bait construct (pMET7-GAG-bait), 0.8 μg prey construct (pMET7-E-tag prey or pMET7-FLAG-Raf), 0.7 μg pMD2.G and 1.4 μg pcDNA3-FLAG-VSV-G. Following overnight transfection, cells were washed once with PBS and 1 ml of fresh growth medium was added to the wells. Cellular debris was removed from the harvested supernatant by 3 minutes centrifugation at 2000×g. The cleared medium was then incubated with 10 μl DYNABEADS® MyOne™ Streptavidin T1 beads (Invitrogen) pre-loaded with 1 μg monoclonal ANTI-FLAG® BioM2-Biotin, Clone M2 (Sigma-Aldrich®) according to the manufacturer&#39;s protocol. After 2 hours binding at 4° C. by end-over-end rotation, beads were washed two times with washing buffer (20 mM HEPES pH 7.4, 150 mM NaCl), and the captured particles were released directly in 35 μl 2×SDS-PAGE loading buffer. A 5-minute incubation step at 65° C. before removal of the beads ensured complete release. After boiling, the samples were loaded on a 10% SDS-PAGE gel, or on commercial 4-12% gradient gels (Biorad), and after separation, the proteins were transferred to HYBOND®-C Extra nitrocellulose membranes (GE Healthcare). Lysates of the producer cells were prepared by direct addition of 200 μl RIPA buffer (50 mM TRIS.HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS+Complete protease inhibitor cocktail [Roche]) to the six-well plates after washing of the cells in chilled PBS. The lysates were cleared by centrifugation at 13000×g, 4° C. for 15 minutes to remove the insoluble fraction. 
         [0033]    The PRS and RRS were randomized and processed in sets of about 45 single PPI measurements. Each set was loaded on two 4-12% gradient gels with 26 slots (Biorad). Each set of measurements also contained the GAG-EGFP expression control, a mock control and the interaction between GRAP2 and LCP2 as a positive control for Virotrap functionality. A single pooled positive control for the GRAP2-LCP2 interaction was also loaded on each gel to allow cross-comparison between the gels. Bands were quantified by fluorescence signals with an ODYSSEY® system (LICOR). The detection threshold was based on RRS signals and was determined for each individual gel. 
         [0034]    For mass spectrometry, 4.75×10 6  HEK293T cells seeded in 75 cm 2  bottles were transfected the next day with a total of 50 μg DNA. The following DNA quantities were used: GAG-bait 25 μg; mock vector 17.8 μg; 7.2 μg of a 50/50 pMD2.G-pcDNA3-FLAG-VSV-G mix. The cellular supernatant was harvested after 32 hours and was centrifuged for 3 minutes at 450×g to remove cellular debris. The cleared supernatant was then filtered using 0.45 μm filters (MILLIPORE®). A total of 100 μl MyOne™ Streptavidin T1 beads pre-loaded with 10 μl ANTI-FLAG® BioM2-Biotin antibody was used to bind the tagged particles. Particles were allowed to bind for 2 hours by end-over-end rotation. Bead-particle complexes were washed once with washing buffer and were then frozen overnight in lysis buffer (PBS, 0.4% CHAPS, 1.2 M guanidine hydrochloride) to release and denature the trapped proteins. After lysis of the particles and removal of the beads, proteins were reduced (10 mM TCEP.HCl, 10 minutes at 37° C.) and alkylated (20 mM Iodoacetamide, 10 minutes at 37° C.). Via NAP5 gel filtration columns (GE Healthcare), the protein sample was transferred to 10 mM ammonium bicarbonate buffer. Trypsin digest was performed overnight at 37° C. using 0.5 μg sequence-grade trypsin (Promega). Samples were vacuum dried and resuspended in 2% acetonitrile, separated by nano-LC and directly analyzed with a LTQ®ORBITRAP® Velos instrument (Thermo Scientific). Searches were performed using the MASCOT® algorithm at 99% confidence against the human Swissprot database complemented with HIV-1 and EGFP protein sequences. 
       Example 1 
     Generation of the Conditional Trapping Construct 
       [0035]    The p55 GAG fusion constructs were generated by PCR amplification using primers Oligo1 and Oligo2 (see Table 1) of the p55 GAG coding sequence from the pCMV-dR8.74 packaging construct (Addgene) and by subsequent IN-FUSION® reaction (Clontech) in a pMET7-gp130-RAS construct (Eyckerman et al., 2001). This resulted in a p55 GAG-fusion construct under control of the strong SRalpha promoter. The plasmid was designated pMET7-GAG-RAS. EGFP was transferred from pEGFP-C1 vector (Clontech) to generate the pMET7-GAG-EGFP construct. The eDHFR fragment was amplified from plasmid pSEL1-eDHFR (Caligiuri et al., 2006) with primers Oligo3 and Oligo4, digested with XhoI and XbaI and cloned in the SalI-XbaI opened pMET7-GAG-RAS backbone, which resulted in pMET7-GAG-eDHFR. The FKBP12 protein was amplified with primers Oligo5 and Oligo6 from pMG2-FKBP12 (Eyckerman et al., 2005). The PCR product was digested with NdeI and XbaI and cloned in the NdeI-XbaI opened pMET7-GAG-eDHFR vector, which resulted in the pMET7-GAG-eDHFR-FKBP12-MCS construct. The reverse primer Oligo6 also encoded a flexible Gly-Gly-Ser hinge sequence and contained a number of restriction enzyme recognition sites. This multi-cloning site (MCS) allows different cloning strategies for the C-terminal fusion of a bait protein. The primers Oligo7 and Oligo8 were annealed and ligated into the NdeI-MluI opened pMET7-GAG-eDHFR-FKBP12-MCS construct to insert a FLAG tag sequence and a T2A auto-processing site. This resulted in pMET7-GAG-eDHFR-T2A-FLAG-FKBP12-MCS. The Thosae asigna 2A (T2A) auto-processing sequence ensures, by a ribosomal skip mechanism (Szymczak et al., 2004), the complete cleavage of the fusion protein resulting in two protein fragments upon translation: the GAG-eDHFR part and the FKBP12-MCS part. The EcoRI site that was present within the eDHFR coding sequence was removed by using site-directed mutagenesis (QUICKCHANGE™ Site-Directed Mutagenesis kit, Stratagene) with Oligo9 and Oligo10 on pMET7-GAG-eDHFR-T2A-FLAG-FKBP12-MCS, resulting in the pMET7-VT1-MCS construct. 
         [0036]    The GATEWAY® cassette (Invitrogen) was amplified by primers Oligo11 and Oligo12 from pMG1-Gateway (Braun et al., 2009), and cloned via MfeI-XbaI in the EcoRI-XbaI opened pMET7-VT1-MCS plasmid, which resulted in the pMET7-VT1-GW destination vector. The coding sequence for CSK1B was transferred via the GATEWAY® LR reaction from the Positive Reference Set described in Braun et al. (Braun et al., 2009) into the pMET7-VT1-GW destination vector resulting in pMET7-VT1-CSK1B. The coding sequence for CDK2 was transferred by the LR reaction to a pMET7-Etag-GATEWAYS construct (Lievens et al., unpublished) leading to pMET7-Etag-CDK2. 
         [0037]    The pcDNA3-FLAG-VSV-G construct used for purification was generated as described (Eyckerman et al., submitted). The pMD2.G construct expressing VSV-G under control of a strong CMV promoter was provided by Didier Trono (EPFL, Lausanne, Switzerland). 
         [0038]    The chemical bivalent molecule or dimerizer consists out of methotrexate (Mtx) and FK506 linked via a PolyEthylene Glycol (PEG) linker, and was prepared as described in Caligiuri et al., 2006. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Oligonucleotides used for the generation of the  
               
               
                 pMET7-VT1 constructs. 
               
             
          
           
               
                   
                   
                   
                 SEQ ID 
               
               
                 Number 
                 Sequence 
                 Use 
                 NO 
               
               
                   
               
             
          
           
               
                 Oligo1 
                 CTCTAAAAGCTGCGGGGCCCGCTAGCGCC 
                 GAG amplification 
                 1 
               
               
                   
                 ACCATGGGTGCGAGAGCGTCAG 
                   
                   
               
               
                   
               
               
                 Oligo2 
                 TGTATTCGGTGAATTCTGAGCTCGTCGAC 
                 GAG amplification 
                 2 
               
               
                   
                 CCGCCTTGTGACGAGGGGTCGCTGC 
                   
                   
               
               
                   
               
               
                 Oligo3 
                 GCGACTCGAGCGGAATCAGTCTGATTGCG 
                 eDHFR 
                 3 
               
               
                   
                 G 
                 amplification 
                   
               
               
                   
               
               
                 Oligo4 
                 CGCTTCTAGATTACATATGGCCGCTGCCC 
                 eDHFR 
                 4 
               
               
                   
                 CGCCGCTCCAGAATCTC 
                 amplification 
                   
               
               
                   
               
               
                 Oligo5 
                 GCGACATATGGGCACGCGTGTGCAGGTG 
                 FKBP12 
                 5 
               
               
                   
                 GAAACCATCTC 
                 amplification 
                   
               
               
                   
               
               
                 Oligo6 
                 CGCTTCTAGATTACTCGAGTGCGGCCGCG 
                 FKBP12 
                 6 
               
               
                   
                 AATTCTGAGCTCGTCGACCCGCCTTCCAG 
                 amplification 
                   
               
               
                   
                 TTTTAGAAGCTCC 
                   
                   
               
               
                   
               
               
                 Oligo7 
                 TATGGAGGGCAGAGGCAGCCTGCTGACCT 
                 T2A and FLAG 
                 7 
               
               
                   
                 GCGGCGACGTGGAGGAAAACCCCGGCCC 
                 sequence annealing 
                   
               
               
                   
                 CGATTACAAGGATGACGACGATAAGA 
                   
                   
               
               
                   
               
               
                 Oligo8 
                 CGCGTCTTATCGTCGTCATCCTTGTAATCG 
                 T2A and FLAG 
                 8 
               
               
                   
                 GGGCCGGGGTTTTCCTCCACGTCGCCGCA 
                 sequence annealing 
                   
               
               
                   
                 GGTCAGCAGGCTGCCTCTGCCCTCCA 
                   
                   
               
               
                   
               
               
                 Oligo9 
                 GAATCGGTATTCAGCGAGTTCCACGATGC 
                 EcoRI mutagenesis 
                 9 
               
               
                   
                 TGATG 
                   
                   
               
               
                   
               
               
                 Oligo10 
                 CATCAGCATCGTGGAACTCGCTGAATACC 
                 EcoRI mutagenesis 
                 10 
               
               
                   
                 GATTC 
                   
                   
               
               
                   
               
               
                 Oligo11 
                 CCCCAATTGACAAGTTTGTACAAAAAAGC 
                 GATEWAY ® 
                 11 
               
               
                   
                   
                 cassette 
                   
               
               
                   
                   
                 amplification 
                   
               
               
                   
               
               
                 Oligo12  
                 GGGTCTAGATCAAACCACTTTGTACAAG 
                 GATEWAY ® 
                 12 
               
               
                   
                   
                 cassette 
                   
               
               
                   
                   
                 amplification 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Production, Harvest and Western Blot Analysis 
       [0039]    For production of virotrap particles, a co-transfection in HEK293T cells was performed via the Ca-Phosphate precipitation method. HEK293T cells were cultured in DMEM medium (Gibco) and 10% FCS at 37° C. in a humidified atmosphere with 5% CO 2 . The day before transfection, 650,000 cells were seeded in a six-well plate. On the day of transfection, a DNA mixture was prepared containing the following:
       0.7 μg pMD2.G   1.4 μg pcDNA3-FLAG-VSV-G   0.8 μg pMET7-Etag-CDK2   3.5 μg pMET7-VT1-CKS1B or pMET7-VT1-EGFP   15 μl of 2.5 M CaCl 2      Water was added to a total volume of 150 μl.       
 
         [0046]    The DNA mixture was then added dropwise to 150 μl 2×HeBs solution while vortexing. The transfection mix was brought on the cells and the precipitates were left overnight for transfection. Following transfection, the cells were washed once with PBS and 1 ml of fresh growth medium was added with either 5 or 10 μM of dimerizer, or without dimerizer. The production medium was harvested after 24 hours. Cellular debris was removed by one minute centrifugation at 2000×g. The cleared medium was then incubated with 10 μl DYNABEADS® MyOne™ Streptavidin T1 beads (Invitrogen) loaded with 1 μg monoclonal ANTI-FLAG® BioM2-Biotin, Clone M2 (Sigma-Aldrich®) according to the manufacturer&#39;s protocol. After 2 hours binding at 4° C. by end-over-end rotation, beads were washed two times with washing buffer (20 mM HEPES pH 7.4, 150 mM NaCl), and the captured nanoparticles were released directly in 35 μl 2×SDS-PAGE loading buffer. The samples were incubated for 5 minutes at 65° C. to ensure complete denaturation/release of the virotrap particles. After removal of the beads and boiling, the samples were loaded on a 10% SDS-PAGE gel, and after separation, the proteins were transferred to HYBOND®-C Extra nitrocellulose membranes (GE Healthcare). Lysates of the producer cells were prepared by direct addition of 200 μl RIPA buffer (50 mM TRIS.HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS+Complete protease inhibitor cocktail [Roche]) to the six-well plates after washing of the cells in chilled PBS. The lysates were cleared by centrifugation at 13000×g, 4° C. for 15 minutes to remove the insoluble fraction. Western blots were probed with mouse anti-E tag (Phadia, 1/1000), mouse anti-GAG (Abcam, 1/1000) or rabbit anti-VSV-G (Sigma/Aldrich, 1/5000). Secondary antibodies were from LI-COR, and blots were digitally imaged using an ODYSSEY® Imager system (LI-COR). 
       Example 3 
     The pMET7-VT1 Construct 
       [0047]    First, a plasmid for expression of the bait construct was generated. The p55 GAG fragment was cloned in the pMET7 vector, which drives expression via the strong SRα promoter. The  E. coli -derived Dihydrofolate reductase (DHFR) protein was fused C-terminally of GAG while the FK506-Binding Protein 12 (FKBP12) coding sequence was cloned via a T2A auto-processing site and a FLAG-tag, in frame and C-terminal of DHFR. The GAG-eDHFR-T2A-FKBP12 expression construct was followed by a multi-cloning site to allow efficient transfer of bait proteins into this expression vector ( FIG. 2 ). 
         [0048]    A GATEWAY® cassette and an EGFP expression construct were inserted in the MCS of pMET7-VT1-MCS by standard cloning procedures. The CSK1B protein was transferred via the GATEWAY® LR reaction into the pMET7-VT1-GATEWAY® construct. 
       Example 4 
     Test of the CKS1B-CDK2 Interaction 
       [0049]    A well-described protein-protein interaction pair was used to test the conditional trapping in nanoparticles. The coding sequence for the CDC28 Protein Kinase Regulatory Subunit 1B (CKS1B) protein was fused to the FKBP12 in the VT1 vector. Addition of the chimeric dimerizer molecule, which consists of methotrexate (Mtx) and FK506 linked by a polyethylene glycol linker, would thus result in the recruitment of the CKS1B bait protein to the GAG protein and to the forming nanoparticles ( FIG. 3 ). The pMET7-VT1-EGFP bait construct was also transfected as a control for irrelevant associations. The interaction partner Cyclin-Dependent Kinase 2 (CDK2), which has an N-terminal E-tag sequence, was co-expressed with both the CSK1B bait protein and the EGFP control protein. Three separate transfections were performed for each bait construct in combination with the CDK2 prey. The first series was left untreated to verify dimerizer-independent interactions, while the second and third series were treated with 5 and 10 μM of dimerizer, respectively. After enrichment and direct elution in SDS loading buffer, the samples were loaded on a 10% PAGE gel and transferred to nitrocellulose membranes after migration. The membranes were first probed with antibodies directed against the E-tag revealing presence of the prey protein in dimerizer-treated and bait-specific conditions. Expression of GAG and VSV-G was verified by using specific antibodies. The expression of the prey (E-tag), GAG and VSV-G was also monitored in the lysates from the producer cells to ensure equal protein levels. 
         [0050]    Clear dimerizer-specific recruitment of the prey construct to the particles can be shown in case of co-expression of the bait CSK1B and prey CDK2. Some weak background association independent from the dimerizer is observed when the EGFP bait is expressed. 
       Example 5 
     Evaluation of the Viral Particle Trap 
       [0051]    To remove the homogenization step in classical AP-MS strategies, it was reasoned that incorporation of a protein complex inside a secreted vesicle should “trap” the interactions under native conditions and should protect the complex during the downstream purification process. As expression of the HIV 1 p55 GAG protein allowed the formation of secreted particles, the concept of packaged or “wrapped” protein complexes was explored by the generation of a plasmid for the expression of GAG fused in frame to a bait protein. A flexible hinge sequence was inserted to limit sterical interference.  FIG. 4 , Panel A shows a schematic presentation of the Virotrap concept. The N-terminus of GAG is essential for membrane association through myristoylation and should thus remain available (Bryant and Ratner, 1990). All domains required for multimerization were still present in the expression construct. As a first PPI pair to evaluate the concept, the H-Ras protein that lacked the myristoylation signal as a bait was selected, combined with the cRAF prey protein. A GAG-EGFP construct and an IRS2 prey were used as irrelevant bait and prey, respectively. Both preys contained an N-terminal FLAG tag to facilitate detection. A first method of particle enrichment was ultracentrifugation, a well-described strategy for the concentration of lentiviral particles for various cell biological applications. After co-expression of bait and prey proteins, cell supernatants were harvested, filtered and centrifuged. After removal of the supernatant, the pelleted particles were resuspended directly in loading buffer and loaded for SDS-PAGE. After Western blotting and revelation of the tagged cRAF protein, clear enrichment of the prey protein could be demonstrated only when the H-Ras bait protein was present ( FIG. 4 , Panels B and C). Expression controls for the particles and the producer cell lysates showed comparable expression for all bait and prey constructs. These results showed that the interaction between H-Ras and cRAF can be detected by co-packaging of bait and prey in secreted particles from live cells. A “mock” empty vector for bait expression was also employed to exclude enrichment of the prey in exosomes that were also pelleted by the ultracentrifugation procedure. 
         [0052]    To remove the tedious ultracentrifugation step, a single-step enrichment protocol for particles in the supernatant was developed and optimized. Briefly, co-expression of the classical VSV-G pseudotyping construct, together with a tagged variant of this glycoprotein, resulted in optimal presentation of the purification tag on the surface of the particles. Paramagnetic beads containing immune reagents for the affinity tag were then employed to capture the particles from the supernatant. First, the interaction between H-Ras and cRAF was confirmed using this new purification strategy. In this case, the system was based on the co-expression of untagged VSV-G with E-tagged VSV-G for capture and purification. HEK293T cells were transfected in a six-well format with GAG-RAS bait together with FLAG-RAF prey, and the controls used in the ultracentrifugation experiment. Virotrap particles were produced for 24 hours and were harvested in 1 ml of supernatant. After purification using anti-E antibodies coupled to paramagnetic particles, SDS-page and Western blotting, the preys were revealed using ANTI-FLAG® antibodies. Clear and specific enrichment of cRAF-prey was shown for the H-Ras-bait protein. No, or very little, cRAF was revealed in case of a-specific bait, while no detectable irrelevant IRS2-prey was found for the H-Ras-bait. 
         [0053]    The interaction between two protein pairs in both directions was then explored. These protein interactions were selected based on literature evidence on their confirmation in independent methods (Braun et al., 2009), and because both protein partners reside in the cytoplasm. In this case, the purification strategy with a FLAG-tagged VSV-G variant was used to replace the E-tagged VSV-G glycoprotein in the previous experiment. After purification by the one-step protocol using ANTI-FLAG® resin, direct elution in PAGE loading buffer, and Western blotting for the E-tag, interactions between CDK2 and CKS1B and between S100A and S100B were readily detected. By swapping bait and prey, the interactions in both directions were shown ( FIG. 4 , Panel D). As controls, irrelevant bait and prey constructs were used. 
         [0054]    By design, the Virotrap system is ideally suited to study cytoplasmic interactions. To explore the uses and limitations and to compare to other existing technologies, the concept was evaluated by testing the human positive reference set (hsPRS-v1). The positive reference set consists of 92 PPIs that were selected based on literature data, while the random reference set is generated using 188 randomly selected proteins (hsRRS-v1; (Venkatesan et al., 2009). Both sets contain proteins from all cellular compartments to remove any bias in localization. All PPIs from the PRS were tested in the Virotrap technology by recombination-assisted transfer of one bait set in fusion to the GAG protein. Prey ORFs were transferred to an expression vector resulting in N-terminally E-tagged fusion proteins. The PPIs were tested in a single direction implying no swap of bait and prey constructs. The same strategy was used for the RRS set, again without swapping of bait and prey proteins. All experiments were performed by transfection of bait and prey expression vectors in HEK293T cells in a six-well format. One day after transfection, supernatants were harvested and processed using the one-step purification protocol. Enriched particles were eluted from the paramagnetic beads in PAGE loading buffer and loaded on SDS-PAGE gels. A total of about 184 binary virotrap experiments were performed. Apart from positive and negative controls, the experiments were controlled for transfection and for immunoblotting efficiency. The presence of prey proteins in purified particles was revealed via anti-E tag immunoblots. The threshold of detection of true positives versus false positives was set for every individual gel. This led to the detection of 28 (31%) interactions in the PRS, while five (5%) interactions were detected in the RRS. Expression of the bait protein fusions in the lysates of the producer cells was verified, which showed that approximately 30% (56 out of 184 bait fusions) was not expressed at a detectable level. Although this could be explained by structural constraints in the fusion proteins or by interference with particle formation, only detectable expression was observed for 25 out of 41 tested prey proteins (61%) of the prey proteins in the producer lysates, where only an N-terminal E-tag was inserted before the protein. Therefore, it is believed that the current data provides an underestimate of the detectable interactions.  FIG. 5  shows the overlap between the Virotrap data and data obtained with other PPI methods for the PRS and RRS as published by Braun and colleagues (2009). Seven interactions out of the positive reference set can be detected with all methods, while nine interactions are unique for the Virotrap method, proving the unique application window for the technology. 
       Example 6 
     Discovery of Novel Interaction Partners Using Co-Complex Virotrap 
       [0055]    For the detection of novel interaction partners, the purification procedure was scaled up to compensate for a larger production scale. 
         [0056]    The protein complexes of two cytosolic proteins were investigated in more detail: Cyclin-Dependent Kinase 2 (CDK2) and Fas-Associated via Death Domain (FADD). An important issue in typical MS co-complex strategies relates to the background. The background in Virotrap will contain GAG- and VSV-G-derived peptide sequences, together with host binding partners, proteins implicated in budding, and serum proteins associated with the outside of the VLPs. To define these background proteins, additional experiments in the design of this study were included to allow the construction of a comprehensive background list. In its simplest format, this list can be subtracted from the protein identifications that are specific for the bait ( FIG. 6 , Panel a). A total of nine experiments were performed (mock control, EGFP bait and five additional bait constructs). Cells were co-transfected with bait proteins and constructs for purification. After harvest and purification, particles were lysed directly on the beads using a chaotropic lysis buffer. The lysates were then processed by reduction and alkylation, buffer exchange and trypsin digest. MS analysis followed by identification via MASCOT (99% confidence) resulted in the identification of between 140 (for LCP2 bait) to 277 (FADD) proteins by at least two unique peptides (Table 2). By comparing the identification lists, a background list of 306 proteins that were found in at least two of the lists were extracted for the different experiments (Table 3). By subtraction of this list from the CDK2 identification list, a limited set of 15 putative binding partners remains. Further removal of protein identifications that were also found with a single peptide in other experiments revealed a short list of seven binding partners. For four of these candidates, there is clear evidence in literature ( FIG. 6 , Panel a). The list of unique proteins for FADD is more extensive (73 proteins), even after removal of proteins that were additionally found in one of the other experiments with a single peptide (35 proteins, Table 4). It is clear that this list contains real binding partners (Receptor interacting protein 1 RIPK1, Casein Kinase 1 alpha and epsilon) as well as unlikely binders. Therefore, the analysis of FADD using additional Virotrap experiments was extended by using a specific elution protocol. In these experiments, the particles from the FLAG-antibody beads were eluted by competition with FLAG-peptide. The particles were then lysed in SDS, processed with detergent removal columns, and digested by trypsin. Controls included in these experiments were mock, EGFP bait and an expression construct of a GAG variant without a bait. Three additional experiments for the FADD interactome were performed ( FIG. 6 , Panel b, Table 5 for overview). Combination of the identifications from these experiments and the previous nine Virotrap assays resulted in a specific list of three candidate partners identified uniquely with at least two peptides in all four FADD experiments: Syndecan 4 (SDC4), Casein kinase 1 epsilon (CSNK1E) and RIPK1. The list of candidate partners identified in two out of four experiments also contained two additional kinases: YES1 and Cyclin-Dependent Kinase 1 (CDK1) ( FIG. 6 , Panel b, right side table). 
         [0057]    Relaxing the criteria where all identifications (including single peptide identifications) in fewer of the repeat samples were included, revealed FAS receptor as candidate interaction partner in the lists of identifications, while TRADD was also found in a single FADD experiment. A20 (TNFAIP3) was identified in the first experiment series, and could be confirmed upon treatment of the cells with TNFα during Virotrap particle production. The interaction between FADD and A20 was also shown by orthogonal co-immunoprecipitation (Co-IP) experiments ( FIG. 6 , Panel c). 
         [0000]    
       
         
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Overview of the proteomics data obtained for different GAG-bait constructs. 
               
               
                 Results were obtained by searches of MS/MS data against all human and bovine 
               
               
                 SWISSPROT accessions, complemented with HIV-1, EGFP and VSV-G sequences by using 
               
               
                 MASCOT ® software. False discovery rates (FDRs) were determined by MASCOT ® searches 
               
               
                 against a database containing all sequences after reversion. Numbers are shown for all identified 
               
               
                 proteins (all) or proteins identified with at least two peptides (&gt;1 peptide). Unique proteins were 
               
               
                 obtained by only considering protein identifications with at least two peptides after removal of 
               
               
                 proteins that were identified in one of the other Virotrap experiments. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 List of background proteins that were found in at least two out  
               
               
                 of nine Virotrap experiments. Both SWISSPROT accessions 
               
               
                 and gene names are shown, as well as the number of 
               
               
                 Virotrap experiments containing the protein (x/9). 
               
             
          
           
               
                   
                   
                 Times 
               
               
                 accession 
                 Gene symbol 
                 identified 
               
               
                   
               
             
          
           
               
                 A5A3E0 
                 POTEF 
                 9 
               
               
                 A5PJE3 
                 FGA 
                 9 
               
               
                 E1B7N2 
                 LOC619094 
                 9 
               
               
                 E1B8G9 
                 HIST3H2BB 
                 9 
               
               
                 E1B953 
                 TUBB 
                 9 
               
               
                 E1B9F6 
                 LOC100848359 
                 9 
               
               
                 E1B9K1 
                 UBC 
                 9 
               
               
                 E1BH06 
                 LOC617696 
                 9 
               
               
                 E1BJB1 
                 TUBB2A 
                 9 
               
               
                 F1MI18 
                 LOC506828 
                 9 
               
               
                 F1MNF8 
                 LOC100141266 
                 9 
               
               
                 F1MNW4 
                 ITIH2 
                 9 
               
               
                 F1MRD0 
                 ACTB 
                 9 
               
               
                 F1MSZ6 
                 SERPINC1 
                 9 
               
               
                 F1MYN5 
                 FBLN1 
                 9 
               
               
                 F1N5M2 
                 GC 
                 9 
               
               
                 G3N2V5 
                 HSP90AB1 
                 9 
               
               
                 G3X6N3 
                 TF 
                 9 
               
               
                 G5E513 
                 Bt.12809 
                 9 
               
               
                 O00560 
                 SDCBP 
                 9 
               
               
                 O46375 
                 TTR 
                 9 
               
               
                 P00735 
                 F2 
                 9 
               
               
                 P01966 
                 HBA 
                 9 
               
               
                 P02081 
                 HBBF_BOVIN Hemoglobin fetal subunit beta 
                 9 
               
               
                   
                 OS = Bos taurus PE = 1 SV = 1 
                   
               
               
                 P02769 
                 ALB 
                 9 
               
               
                 P07437 
                 TUBB 
                 9 
               
               
                 P07900 
                 HSP90AA1 
                 9 
               
               
                 P08107 
                 HSPA1A 
                 9 
               
               
                 P08670 
                 VIM 
                 9 
               
               
                 P11142 
                 HSPA8 
                 9 
               
               
                 P12268 
                 IMPDH2 
                 9 
               
               
                 P12763 
                 AHSG 
                 9 
               
               
                 P15497 
                 APOA1 
                 9 
               
               
                 P22626 
                 HNRNPA2B1 
                 9 
               
               
                 P28800 
                 SERPINF2 
                 9 
               
               
                 P34955 
                 SERPINA1 
                 9 
               
               
                 P35580 
                 MYH10 
                 9 
               
               
                 P56652 
                 ITIH3 
                 9 
               
               
                 P81187 
                 CFB 
                 9 
               
               
                 P81644 
                 APOA2 
                 9 
               
               
                 Q00839 
                 HNRNPU 
                 9 
               
               
                 Q03247 
                 APOE 
                 9 
               
               
                 Q07020 
                 RPL18 
                 9 
               
               
                 Q08431 
                 MFGE8 
                 9 
               
               
                 Q13813 
                 SPTAN1 
                 9 
               
               
                 Q3SZ57 
                 AFP 
                 9 
               
               
                 Q3SZR3 
                 ORM1 
                 9 
               
               
                 Q3ZBS7 
                 VTN 
                 9 
               
               
                 Q58D62 
                 FETUB 
                 9 
               
               
                 Q7SIH1 
                 A2M 
                 9 
               
               
                 Q9BQA1 
                 WDR77 
                 9 
               
               
                 A2I7M9 
                 SERPINA3-2 
                 8 
               
               
                 F1MGU7 
                 FGG 
                 8 
               
               
                 F1MMK9 
                 AMBP 
                 8 
               
               
                 Q29443 
                 TF 
                 8 
               
               
                 Q2KJF1 
                 A1BG 
                 8 
               
               
                 Q3SZV7 
                 HPX 
                 8 
               
               
                 F1MBQ8 
                 DDX5 
                 8 
               
               
                 F2Z4C1 
                 TUBA3C 
                 8 
               
               
                 G5E507 
                 HSP90AB1 
                 8 
               
               
                 P17697 
                 CLU 
                 8 
               
               
                 P35527 
                 KRT9 
                 8 
               
               
                 P60709 
                 ACTB 
                 8 
               
               
                 Q3SYW6 
                 EIF3C 
                 8 
               
               
                 A1A4R1 
                 HIST2H2AC 
                 8 
               
               
                 P01045 
                 KNG2 
                 8 
               
               
                 P26373 
                 RPL13 
                 8 
               
               
                 12831136 
                 gb|AAK08483.1|AF324493_2 gag polyprotein 
                 8 
               
               
                   
                 [HIV-1 vector pNL4-3] 
                   
               
               
                 A6NKZ8 
                 YI016_HUMAN Putative tubulin beta chain-like 
                 8 
               
               
                   
                 protein ENSP00000290377 OS =  Homo sapiens   
                   
               
               
                   
                 PE = 5 SV = 2 
                   
               
               
                 Q28107 
                 F5 
                 8 
               
               
                 P04264 
                 KRT1 
                 8 
               
               
                 P13645 
                 KRT10 
                 8 
               
               
                 A6QLG5 
                 RPS9 
                 8 
               
               
                 A7E350 
                 PLG 
                 8 
               
               
                 E1BEG2 
                 HNRNPA3 
                 8 
               
               
                 G8JKY0 
                 RPS8 
                 8 
               
               
                 P15311 
                 EZR 
                 8 
               
               
                 P39023 
                 RPL3 
                 8 
               
               
                 P62424 
                 RPL7A 
                 8 
               
               
                 A2I7N3 
                 SERPINA3-7 
                 7 
               
               
                 A7E307 
                 DDX17 
                 7 
               
               
                 F1MJH1 
                 GSN 
                 7 
               
               
                 F1MY44 
                 HNRNPM 
                 7 
               
               
                 G1K122 
                 RBP4 
                 7 
               
               
                 O43175 
                 PHGDH 
                 7 
               
               
                 P00003 
                 FLAG-VSVG 
                 7 
               
               
                 P12259 
                 F5 
                 7 
               
               
                 P61978 
                 HNRNPK 
                 7 
               
               
                 Q9TTJ5 
                 RGN 
                 7 
               
               
                 Q3Y5Z3 
                 ADIPOQ 
                 7 
               
               
                 E1BF20 
                 HNRNPH1 
                 7 
               
               
                 G5E5T5 
                 G5E5T5_BOVIN Uncharacterized protein 
                 7 
               
               
                   
                 (Fragment) OS = Bos taurus PE = 4 SV = 1 
                   
               
               
                 296556483 
                 gb|AAK08484.2|AF324493_3 pol polyprotein 
                 7 
               
               
                   
                 [HIV-1 vector pNL4-3] 
                   
               
               
                 E1B7J1 
                 E1B7J1_BOVIN Elongation factor 1-alpha 
                 7 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 E1BAK6 
                 DAZAP1 
                 7 
               
               
                 F1MWU9 
                 HSPA6 
                 7 
               
               
                 P40429 
                 RPL13A 
                 7 
               
               
                 A5D9B4 
                 HNRPH2 
                 7 
               
               
                 E1BHA5 
                 E1BHA5_BOVIN Uncharacterized protein 
                 7 
               
               
                   
                 OS = Bos taurus PE = 4 SV = 1 
                   
               
               
                 P09651 
                 HNRNPA1 
                 7 
               
               
                 P11940 
                 PABPC1 
                 7 
               
               
                 P61353 
                 RPL27 
                 7 
               
               
                 A2VE06 
                 RPS4Y1 
                 7 
               
               
                 G3N262 
                 G3N262 BOVIN Uncharacterized protein 
                 7 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 P62269 
                 RPS18 
                 7 
               
               
                 P26038 
                 MSN 
                 7 
               
               
                 A7YW45 
                 PRMT5 
                 6 
               
               
                 G3MYZ3 
                 AFM 
                 6 
               
               
                 Q05443 
                 LUM 
                 6 
               
               
                 F1MY85 
                 C5 
                 6 
               
               
                 A6NHL2 
                 TUBAL3 
                 6 
               
               
                 P60842 
                 EIF4A1 
                 6 
               
               
                 F1MQ37 
                 MYH9 
                 6 
               
               
                 A6QPP2 
                 SERPIND1 
                 6 
               
               
                 P35579 
                 MYH9 
                 6 
               
               
                 P35908 
                 KRT2 
                 6 
               
               
                 P29966 
                 MARCKS 
                 6 
               
               
                 E1B7R4 
                 EIF3A 
                 6 
               
               
                 P13639 
                 EEF2 
                 6 
               
               
                 P12277 
                 CKB 
                 6 
               
               
                 P23528 
                 CFL1 
                 6 
               
               
                 E1B8G4 
                 E1B8G4_BOVIN Uncharacterized protein 
                 6 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 2 
                   
               
               
                 P62917 
                 RPL8 
                 6 
               
               
                 Q8IX12 
                 CCAR1 
                 6 
               
               
                 P04406 
                 GAPDH 
                 6 
               
               
                 P14618 
                 PKM 
                 6 
               
               
                 Q0VCZ3 
                 YTHDF2 
                 6 
               
               
                 G3MX91 
                 TARDBP 
                 6 
               
               
                 F1MI47 
                 RBM14 
                 5 
               
               
                 A5D784 
                 CPNE8 
                 5 
               
               
                 G3N0S9 
                 LOC515150 
                 5 
               
               
                 F1MNV5 
                 KNG1 
                 5 
               
               
                 Q2UVX4 
                 C3 
                 5 
               
               
                 A5PK20 
                 HIST1H1E 
                 5 
               
               
                 F1MYC9 
                 SPTBN1 
                 5 
               
               
                 F1MVC0 
                 CAD 
                 5 
               
               
                 E1BGR6 
                 E1BGR6_BOVIN Uncharacterized protein 
                 5 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 P62249 
                 RPS16 
                 5 
               
               
                 P83731 
                 RPL24 
                 5 
               
               
                 G3X861 
                 G3X861_BOVIN Uncharacterized protein 
                 5 
               
               
                   
                 (Fragment) OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 P62280 
                 RPS11 
                 5 
               
               
                 P08865 
                 RPSA 
                 5 
               
               
                 A5PKD6 
                 GNB4 
                 5 
               
               
                 F1MKC4 
                 F1MKC4_BOVIN Uncharacterized protein 
                 5 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 2 
                   
               
               
                 P26641 
                 EEF1G 
                 5 
               
               
                 Q06830 
                 PRDX1 
                 5 
               
               
                 P09543 
                 CNP 
                 5 
               
               
                 F1MMD7 
                 ITIH4 
                 5 
               
               
                 Q3T052 
                 ITIH4 
                 4 
               
               
                 P02768 
                 ALB 
                 4 
               
               
                 P55884 
                 EIF3B 
                 4 
               
               
                 P02538 
                 KRT6A 
                 4 
               
               
                 A7MAZ5 
                 HIST1H1D 
                 4 
               
               
                 P35613 
                 BSG 
                 4 
               
               
                 Q3SZH5 
                 AGT 
                 4 
               
               
                 P39060 
                 COL18A1 
                 4 
               
               
                 P60033 
                 CD81 
                 4 
               
               
                 P62937 
                 PPIA 
                 4 
               
               
                 Q01082 
                 SPTBN1 
                 4 
               
               
                 Q08E32 
                 CHMP4B 
                 4 
               
               
                 A5PK61 
                 H3F3C 
                 4 
               
               
                 Q13151 
                 HNRNPA0 
                 4 
               
               
                 F1MB60 
                 RPS26 
                 4 
               
               
                 P23396 
                 RPS3 
                 4 
               
               
                 F1MMP5 
                 ITIH1 
                 4 
               
               
                 O15372 
                 EIF3H 
                 4 
               
               
                 P46777 
                 RPL5 
                 4 
               
               
                 Q02543 
                 RPL18A 
                 4 
               
               
                 G3X7A5 
                 C3 
                 4 
               
               
                 E1BE42 
                 E1BE42_BOVIN Uncharacterized protein 
                 4 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 Q9Y265 
                 RUVBL1 
                 4 
               
               
                 O18789 
                 RPS2 
                 4 
               
               
                 G3N2F0 
                 G3N2F0_BOVIN Elongation factor 1-alpha 
                 4 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 P62913 
                 RPL11 
                 4 
               
               
                 A6NMY6 
                 ANXA2P2 
                 4 
               
               
                 E1BB17 
                 HNRNPH3 
                 4 
               
               
                 P08779 
                 KRT16 
                 4 
               
               
                 F6QVC9 
                 ANXA5 
                 4 
               
               
                 E1BNB4 
                 PABPC1L 
                 4 
               
               
                 A6H769 
                 RPS7 
                 4 
               
               
                 E1BAT6 
                 E1BAT6_BOVIN Uncharacterized protein 
                 4 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 Q562R1 
                 ACTBL2 
                 4 
               
               
                 Q8WUM4 
                 PDCD6IP 
                 4 
               
               
                 P19338 
                 NCL 
                 3 
               
               
                 Q3SYR0 
                 SERPINA7 
                 3 
               
               
                 P01614 
                 KV201_HUMAN Ig kappa chain V-II region 
                 3 
               
               
                   
                 Cum OS = Homo sapiens PE = 1 SV = 1 
                   
               
               
                 P17690 
                 APOH 
                 3 
               
               
                 G3N361 
                 NONO 
                 3 
               
               
                 P63243 
                 GNB2L1 
                 3 
               
               
                 P84103 
                 SRSF3 
                 3 
               
               
                 Q01130 
                 SRSF2 
                 3 
               
               
                 G8JKV5 
                 RPL14 
                 3 
               
               
                 F1MJM0 
                 ZNF326 
                 3 
               
               
                 F1ML72 
                 RPL34 
                 3 
               
               
                 FlMSD2 
                 RUVBL2 
                 3 
               
               
                 O15371 
                 EIF3D 
                 3 
               
               
                 P08621 
                 SNRNP70 
                 3 
               
               
                 P18621 
                 RPL17 
                 3 
               
               
                 G3X8B1 
                 LOC613401 
                 3 
               
               
                 P62935 
                 PPIA 
                 3 
               
               
                 O43242 
                 PSMD3 
                 3 
               
               
                 P41252 
                 IARS 
                 3 
               
               
                 P49327 
                 FASN 
                 3 
               
               
                 Q3MHL4 
                 AHCY 
                 3 
               
               
                 Q86YQ8 
                 CPNE8 
                 3 
               
               
                 G5E604 
                 G5E604_BOVIN Uncharacterized protein 
                 3 
               
               
                   
                 (Fragment) OS = Bos taurus PE = 4 SV = 1 
                   
               
               
                 Q12906 
                 ILF3 
                 3 
               
               
                 A7MB16 
                 EIF3B 
                 3 
               
               
                 F1MH40 
                 Bt.57604 
                 3 
               
               
                 E1BCL3 
                 LOC507211 
                 3 
               
               
                 P54727 
                 RAD23B 
                 3 
               
               
                 G3N2D7 
                 IGLL1 
                 3 
               
               
                 A7MBG8 
                 RUVBL1 
                 3 
               
               
                 F1MZ00 
                 SNRPD3 
                 3 
               
               
                 F1N6C0 
                 F1N6C0_BOVIN Uncharacterized protein 
                 3 
               
               
                   
                 OS = Bos taurus PE-4 SV = 2 
                   
               
               
                 Q13310 
                 PABPC4 
                 3 
               
               
                 F1MXE4 
                 PSMD6 
                 3 
               
               
                 A4IFP7 
                 ARF5 
                 3 
               
               
                 F1N0E5 
                 CCT4 
                 3 
               
               
                 P46779 
                 RPL28 
                 3 
               
               
                 A5PK39 
                 TPP2 
                 3 
               
               
                 P78371 
                 CCT2 
                 3 
               
               
                 P02786 
                 TFRC 
                 3 
               
               
                 F1MPU0 
                 CLTC 
                 3 
               
               
                 O14744 
                 PRMT5 
                 3 
               
               
                 Q969P0 
                 IGSF8 
                 3 
               
               
                 Q9P2B2 
                 PTGFRN 
                 3 
               
               
                 P07224 
                 PROS1 
                 2 
               
               
                 Q28085 
                 CFH 
                 2 
               
               
                 F1MG05 
                 EEF1G 
                 2 
               
               
                 F1MLW8 
                 LOC100847119 
                 2 
               
               
                 E1BMJ0 
                 LOC100847889 
                 2 
               
               
                 A0JND2 
                 KRT80 
                 2 
               
               
                 B8Y9S9 
                 FN1 
                 2 
               
               
                 P02656 
                 APOC3 
                 2 
               
               
                 Q3MHN2 
                 C9 
                 2 
               
               
                 F6QYV9 
                 SSRP1 
                 2 
               
               
                 P02253 
                 HIST1H1C 
                 2 
               
               
                 P07910 
                 HNRNPC 
                 2 
               
               
                 P08758 
                 ANXA5 
                 2 
               
               
                 P43243 
                 MATR3 
                 2 
               
               
                 Q9Y2W1 
                 THRAP3 
                 2 
               
               
                 E1BQ37 
                 SFPQ 
                 2 
               
               
                 P11586 
                 MTHFD1 
                 2 
               
               
                 A6QLT5 
                 UBAP2L 
                 2 
               
               
                 E1B9M9 
                 L00525863 
                 2 
               
               
                 P20645 
                 M6PR 
                 2 
               
               
                 Q96EP5 
                 DAZAP1 
                 2 
               
               
                 P40227 
                 CCT6A 
                 2 
               
               
                 P61024 
                 CKS1B 
                 2 
               
               
                 G1K134 
                 Bt.57435 
                 2 
               
               
                 P84090 
                 ERH 
                 2 
               
               
                 P30101 
                 PDIA3 
                 2 
               
               
                 F1MZ92 
                 YBX1 
                 2 
               
               
                 A4IFC3 
                 PABPC4 
                 2 
               
               
                 A5PK63 
                 RPS17 
                 2 
               
               
                 E1BCF5 
                 RPL26L1 
                 2 
               
               
                 F1MHJ6 
                 F1MHJ6_BOVIN 60S ribosomal protein 
                 2 
               
               
                   
                 L18a OS = Bos taurus PE = 3 SV = 2 
                   
               
               
                 F1MLH6 
                 CALM2 
                 2 
               
               
                 P05543 
                 SERPINA7 
                 2 
               
               
                 P13010 
                 XRCC5 
                 2 
               
               
                 Q15366 
                 PCBP2 
                 2 
               
               
                 E1BF81 
                 SERPINA6 
                 2 
               
               
                 P16403 
                 HIST1H1C 
                 2 
               
               
                 G5E531 
                 TCP1 
                 2 
               
               
                 P02788 
                 LTF 
                 2 
               
               
                 P08238 
                 HSP90AB1 
                 2 
               
               
                 P62194 
                 PSMC5 
                 2 
               
               
                 P04350 
                 TUBB4A 
                 2 
               
               
                 P02533 
                 KRT14 
                 2 
               
               
                 Q5D862 
                 FLG2 
                 2 
               
               
                 D3IVZ2 
                 DDX3Y 
                 2 
               
               
                 075131 
                 CPNE3 
                 2 
               
               
                 P57721 
                 PCBP3 
                 2 
               
               
                 Q5VW32 
                 BROX 
                 2 
               
               
                 E1BKM4 
                 PDCD6IP 
                 2 
               
               
                 P60660 
                 MYL6 
                 2 
               
               
                 F1MZV2 
                 CHMP5 
                 2 
               
               
                 O75340 
                 PDCD6 
                 2 
               
               
                 P29144 
                 TPP2 
                 2 
               
               
                 A5D9H5 
                 HNRPD 
                 2 
               
               
                 A6NIZ1 
                 RP1BL_HUMAN Ras-related protein Rap-1b- 
                 2 
               
               
                   
                 like protein OS = Homo sapiens PE = 2 SV = 1 
                   
               
               
                 A7Z057 
                 YWHAG 
                 2 
               
               
                 E1B726 
                 PLG 
                 2 
               
               
                 E1B7T4 
                 E1B7T4_BOVIN Uncharacterized protein 
                 2 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 2 
                   
               
               
                 E1BK63 
                 E1BK63_BOVIN Ribosomal protein L15 
                 2 
               
               
                   
                 OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 E1BNR0 
                 Bt.110587 
                 2 
               
               
                 G3MYE2 
                 G3MYE2_BOVIN Uncharacterized protein 
                 2 
               
               
                   
                 (Fragment) OS = Bos taurus PE = 3 SV = 1 
                   
               
               
                 O14828 
                 SCAMP3 
                 2 
               
               
                 P00004 
                 VSVG 
                 2 
               
               
                 P05023 
                 ATP1A1 
                 2 
               
               
                 P06733 
                 ENO1 
                 2 
               
               
                 P08195 
                 SLC3A2 
                 2 
               
               
                 P18124 
                 RPL7 
                 2 
               
               
                 P27635 
                 RPL10 
                 2 
               
               
                 P36578 
                 RPL4 
                 2 
               
               
                 P49006 
                 MARCKSL1 
                 2 
               
               
                 P52272 
                 HNRNPM 
                 2 
               
               
                 P53985 
                 SLC16A1 
                 2 
               
               
                 P61204 
                 ARF3 
                 2 
               
               
                 P62258 
                 YWHAE 
                 2 
               
               
                 P62847 
                 RPS24 
                 2 
               
               
                 Q02878 
                 RPL6 
                 2 
               
               
                 Q14152 
                 EIF3A 
                 2 
               
               
                 Q15758 
                 SLC1A5 
                 2 
               
               
                 Q53EZ4 
                 CEP55 
                 2 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 List of FADD interaction partners. 
               
               
                 List of FADD interaction partners after removal  
               
               
                 of all proteins (including single peptide protein  
               
               
                 identifications) that were found in at least one of the  
               
               
                 other Virotrap experiments. Proteins in bold have 
               
               
                 been linked to FADD or to FAS signaling before. 
               
             
          
           
               
                   
                 Accession 
                 Protein 
               
               
                   
               
             
          
           
               
                 1  
                 A2VDY3 
                 CHMP4A 
               
               
                 2  
                 A4FUC2 
                 HNRNPUL1 
               
               
                 3  
                 A6QLS9 
                 RAB10 
               
               
                 4  
                 E1BAF6 
                 PRRC2A 
               
               
                 5  
                 
                   F1MND1 
                 
                 
                   CDC42 
                 
               
               
                 6  
                 F1MSI2 
                 AGRN 
               
               
                 7  
                 F1MX61 
                 SF3B1 
               
               
                 8  
                 G3N3Q3 
                 G3N3Q3_BOVIN 
               
               
                 9  
                 G5E5V7 
                 G5E5V7_BOVIN 
               
               
                 10 
                 O00232 
                 PSMD12 
               
               
                 11 
                 O00299 
                 CLIC1 
               
               
                 12 
                 O60884 
                 DNAJA2 
               
               
                 13 
                 P01891 
                 HLA-A 
               
               
                 14 
                 P05556 
                 ITGB1 
               
               
                 15  
                 
                   P06493 
                 
                 
                   CDK1 
                 
               
               
                 16 
                 P07195 
                 LDHB 
               
               
                 17 
                 P11017 
                 GNB2 
               
               
                 18 
                 
                   P21580 
                 
                 
                   TNFAIP3 
                 
               
               
                 19 
                 
                   P31431 
                 
                 
                   SDC4 
                 
               
               
                 20 
                 P31689 
                 DNAJA1 
               
               
                 21 
                 P48643 
                 CCT5 
               
               
                 22 
                 
                   P48729 
                 
                 
                   CSNK1A1 
                 
               
               
                 23 
                 
                   P49674 
                 
                 
                   CSNK1E 
                 
               
               
                 24 
                 P60174 
                 TPI1 
               
               
                 25 
                 P61247 
                 RPS3A 
               
               
                 26 
                 P62871 
                 GNB1 
               
               
                 27 
                 P62888 
                 RPL30 
               
               
                 28 
                 
                   Q13158 
                 
                 
                   FADD 
                 
               
               
                 29 
                 
                   Q13546 
                 
                 
                   RIPK1 
                 
               
               
                 30 
                 Q148F1 
                 CFL2 
               
               
                 31 
                 Q8TB73 
                 NDNF 
               
               
                 32 
                 Q99661 
                 KIF2C 
               
               
                 33 
                 Q9NRX5 
                 SERINC1 
               
               
                 34 
                 Q9UN37 
                 VPS4A 
               
               
                 35 
                 Q9Y5K6 
                 CD2AP 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Overview of the proteomics data obtained for different GAG-bait 
               
               
                 constructs using specific elution of particles from the purification beads. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 # Unique 
                   
               
               
                   
                 PRIDE 
                   
                 #  
                 # 
                 # Proteins 
                 proteins 
                   
               
               
                   
                 Experiment 
                 # 
                 Peptide 
                 Proteins 
                 (&gt;1 
                 (&gt;1 
                 FDR 
               
               
                   
                 Accession 
                 Spectra 
                 sequences 
                 (all) 
                 peptide) 
                 peptide) 
                 (%) 
               
               
                   
               
             
          
           
               
                 mock 
                 28963 
                 6706 
                 557 
                 169 
                 95 
                 14 
                 0.6 
               
               
                 sGAG* 
                 28965 
                 7661 
                 595 
                 255 
                 117 
                 18 
                 0.4 
               
               
                 GAG- 
                 28964 
                 6450 
                 397 
                 242 
                 114 
                 29 
                 1.7 
               
               
                 EGFP 
                   
                   
                   
                   
                   
                   
                   
               
               
                 GAG- 
                 28962 
                 7546 
                 864 
                 375 
                 174 
                 20 
                 0.7 
               
               
                 FADD 
                   
                   
                   
                   
                   
                   
                   
               
               
                 GAG- 
                 28960 
                 8377 
                 517 
                 166 
                 98 
                 2 
                 0.4 
               
               
                 FADD 
                   
                   
                   
                   
                   
                   
                   
               
               
                 GAG- 
                 28961 
                 8388 
                 717 
                 231 
                 121 
                 9 
                 0.5 
               
               
                 FADD** 
               
               
                   
               
               
                 Results were obtained by searches of MS data against all human and bovine SWISSPROT accessions, complemented with HIV-1, EGFP and VSV-G sequences. False discovery rates (FDRs) were determined by MASCOT searches against a database containing all sequences after inversion. 
               
               
                 *an alternative codon-optimized GAG construct without a bait was used to generate particles. 
               
               
                 **GAG-FADD Virotrap particles were produced in the presence of TNFα. 
               
             
          
         
       
     
       Example 7 
     Identification of Desmosomal Components 
       [0058]    By employing a similar background removal strategy for the S100A1 bait as for the CDK2 bait (i.e., removal of all a-specific proteins identified) in the first set of nine experiments, a list of ten putative interaction partners identified with at least two peptides was obtained (Table 6). Remarkably, three components of the desmosome can be found in this list (Desmoplakin DSP, Desmoglein 1 DSG1 and Junction Plakoglobin JUP), as well as two keratin proteins not found in other bait proteins (thus, not constituting classical contaminating keratins). These keratins are known intermediate filament components that use the desmosome for anchoring (Kitajima, 2013). The link between S100 proteins and desmosomes is hinted in literature. The S100A10 and S100A11 can be found together with desmosomal proteins in the cornified envelope (Robinson et al., 1997). Various members of the S100 family of proteins have been implicated in inflammatory skin disorders affecting the integrity of the skin such as psoriasis (Eckert et al., 2004). In addition, it is clear that these calcium-binding proteins play an important role in metastasis of tumors, both in the primary tumor cells and the metastatic niche (Lukanidin and Sleeman, 2012). The S100A1 protein also plays an important role in striated muscle and has been implicated in myocardial (dys)function and heart failure (Krause et al., 2009). 
         [0059]      FIG. 7  shows the mapping of the identified peptides of Desmoglein 1 on the amino acid sequence. Peptides from both the extracellular part and the intracellular part of the protein were identified. In Table 7, the identified peptides for Desmoglein 1 are shown with their MASCOT scores. This data clearly supports the fact that Virotrap allows the detection of transmembrane prey proteins. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Putative interaction partners for the S100A1 bait protein. 
               
               
                 Desmosomal or intermediate filament proteins  
               
               
                 are annotated in the comments column. 
               
             
          
           
               
                   
                 Accession 
                 Protein 
                 Comment 
               
               
                   
                   
               
               
                   
                 P05089 
                 ARG1 
                   
               
               
                   
                 P13647 
                 KRT5 
                 Intermediate filament 
               
               
                   
                 P14923 
                 JUP 
                 Desmosome component 
               
               
                   
                 P15924 
                 DSP 
                 Desmosome component 
               
               
                   
                 P23297 
                 S100A1 
                 BAIT 
               
               
                   
                 Q02413 
                 DSG1 
                 Desmosome component 
               
               
                   
                 Q6UWP8 
                 SBSN 
                   
               
               
                   
                 Q8N1N4 
                 KRT78 
                 Intermediary filament 
               
               
                   
                 Q96P63 
                 SERPINB12 
                   
               
               
                   
                 Q99816 
                 TSG101 
                   
               
               
                   
                 Q9UK41 
                 VPS28 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 the identified peptides for Desmoglein 1 (DSG1, Swissprot Acces- 
               
               
                 sion Q02413) are shown with their respective MASCOT ® scores. 
               
             
          
           
               
                   
                 start aa 
                 end aa 
                   
                 MASCOT ® 
                 SEQ ID 
               
               
                 Peptide 
                 position 
                 position 
                 modified peptide sequence 
                 score 
                 NO 
               
               
                   
               
             
          
           
               
                 I 
                 439 
                 445 
                 NH2-TGKLTLK-COOH 
                 33 
                 13 
               
               
                   
               
               
                 E 
                 916 
                 925 
                 NH2-ESSNVVVTER-COOH 
                 77 
                 14 
               
               
                   
               
               
                 J1 
                 326 
                 332 
                 NH2-TNVGILK-COOH 
                 41 
                 15 
               
               
                   
               
               
                 F 
                 198 
                 213 
                 NH2- 
                 57 
                 16 
               
               
                   
                   
                   
                 IIRQEPSDSPM(oxid)FIINR- 
                   
                   
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 A 
                 129 
                 144 
                 NH2-ALNSMGQDLERPLELR- 
                 48 
                 17 
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 M + K 
                 391 
                 422 
                 NH2- 
                 84 
                 18 
               
               
                   
                   
                   
                 TYVVTGNMGSNDKVGDFVAT 
                   
                   
               
               
                   
                   
                   
                 DLDTGRPSTTVR-COOH 
                   
                   
               
               
                   
               
               
                 F 
                 198 
                 213 
                 NH2-IIRQEPSDSPMFIINR- 
                 74 
                 19 
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 D 
                 220 
                 238 
                 NH2- 
                 73 
                 20 
               
               
                   
                   
                   
                 TM(oxid)NNFLDREQYGQYAL 
                   
                   
               
               
                   
                   
                   
                 AVR-COOH 
                   
                   
               
               
                   
               
               
                 L 
                 423 
                 438 
                 NH2-YVMGNNPADLLAVDSR- 
                 107 
                 21 
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 D 
                 220 
                 238 
                 NH2- 
                 75 
                 22 
               
               
                   
                   
                   
                 TMNNFLDREQYGQYALAVR- 
                   
                   
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 J2 
                 333 
                 352 
                 NH2- 
                 49 
                 23 
               
               
                   
                   
                   
                 VVKPLDYEAMQSLQLSIGVR- 
                   
                   
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 G 
                 87 
                 105 
                 NH2-ISGVGIDQPPYGIFVINQK- 
                 118 
                 24 
               
               
                   
                   
                   
                 COOH 
                   
                   
               
               
                   
               
               
                 B 
                 369 
                 390 
                 NH2- 
                 86 
                 25 
               
               
                   
                   
                   
                 ASAISVTVLNVIEGPVFRPGSK- 
                   
                   
               
               
                   
                   
                   
                 COOH 
               
               
                   
               
             
          
         
       
     
       REFERENCES 
       [0000]    
       
         Booher R. N., C. E. Alfa, J. S. Hyams, and D. H. Beach (1989). The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization.  Cell  58:485-497. 
         Braun P., M. Tasan, M. Dreze, M. Barrios-Rodiles, I. Lemmens, H. Yu, J. M. Sahalie, R. R. Murray, L. Roncari, and A. S. de Smet, et al. (2009). An experimentally derived confidence score for binary protein-protein interactions.  Nat. Methods  6:91-97. 
         Briggs J. A., M. N. Simon, I. Gross, H. G. Krausslich, S. D. Fuller, V. M. Vogt, and M. C. Johnson (2004). The stoichiometry of Gag protein in HIV-1 . Nat. Struct. Mol. Biol.  11:672-675. 
         Bryant M. and L. Ratner (1990). Myristoylation-dependent replication and assembly of human immunodeficiency virus 1 . Proc. Natl. Acad. Sci. U.S.A.  87:523-527. 
         Caligiuri M., L. Molz, Q. Liu, F. Kaplan, J. P. Xu, J. Z. Majeti, R. Ramos-Kelsey, K. Murthi, S. Lievens, and J. Tavernier, et al. (2006). MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells.  Chem. Biol.  13:711-722. 
         Eyckerman S., I. Lemmens, D. Catteeuw, A. Verhee, J. Vandekerckhove, S. Lievens, and J. Tavernier (2005). Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.  Nat. Methods  2:427-433. 
         Eyckerman S., A. Verhee, J. V. der Heyden, I. Lemmens, X. V. Ostade, J. Vandekerckhove, and J. Tavernier (2001). Design and application of a cytokine-receptor-based interaction trap.  Nat. Cell. Biol.  3:1114-1119. 
         Eckert R. L., A. M. Broome, M. Ruse, N. Robinson, D. Ryan and K. Lee (2004). S100 proteins in the epidermis.  J. Invest. Dermatol.  123:23-33. 
         Gavin A. C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, J. M. Rick, A. M. Michon, and C. M. Cruciat, et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes.  Nature  415:141-147. 
         Gheysen D., E. Jacobs, F. de Foresta, C. Thiriart, M. Francotte, D. Thines, and M. De Wilde (1989). Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells.  Cell  59:103-112. 
         Gingras A. C., M. Gstaiger, B. Raught, and R. Aebersold (2007). Analysis of protein complexes using mass spectrometry.  Nat. Rev. Mol. Cell. Biol.  8:645-654. 
         Gomez C. Y. and T. J. Hope (2006). Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells.  J. Virol.  80:8796-8806. 
         Kitajima Y. (2013). Regulation and impairments of dynamic desmosome and carneodesmosome remodeling.  Eur. J. Dermatol . April 30, E pub ahead of print. 
         Kraus C., D. Rohde, C. Weidenhammer, G. Qiu, S. T. Pleger, M. Voelkers, M. Boerries, A. Remppis, H. A. Katus, and P. Most (2009). S100A1 in cardiovascular health and disease: closing the gap between basic science and clinical therapy.  J. Mol. Cell. Cardiol.  47:445-455. 
         Lewitzky M., C. Kardinal, N. H. Gehring, E. K. Schmidt, B. Konkol, M. Eulitz, W. Birchmeier, U. Schaeper, and S. M. Feller (2001). The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.  Oncogene  20:1052-1062. 
         Lukanidin E. and J. P. Sleeman (2012). Building the niche: the role of the S100 proteins in metastatic growth.  Seminars in Cancer Biology  22:216-225. 
         Malovannaya A., R. B. Lanz, S. Y. Jung, Y. Bulynko, N. T. Le, D. W. Chan, C. Ding, Y. Shi, N. Yucer, and G. Krenciute, et al. (2011). Analysis of the human endogenous coregulator complexome.  Cell  145:787-799. 
         Nigg E. A. (1995). Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle.  Bioessays  17:471-480. 
         Paul F. E., F. Hosp, and M. Selbach (2011). Analyzing protein-protein interactions by quantitative mass spectrometry.  Methods  54:387-395. 
         Prince T., L. Sun, and R. L. Matts (2005). Cdk2: a genuine protein kinase client of Hsp90 and Cdc37 . Biochemistry  44:15287-15295. 
         Robinson N. A., S. Lapic, J. F. Welter, and R. L. Eckert (1997). S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.  J. Biol. Chem.  272:12035-12046. 
         Shioda T. and H. Shibuta (1990). Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV.  Virology  175:139-148. 
         Szymczak A. L., C. J. Workman, Y. Wang, K. M. Vignali, S. Dilioglou, E. F. Vanin, and D. A. Vignali, D. A. (2004). Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector.  Nat. Biotechnol.  22:589-594. 
         Venkatesan K., J. F. Rual, A. Vazquez, U. Stelzl, I. Lemmens, T. Hirozane-Kishikawa, T. Hao, M. Zenkner, X. Xin, and K. I. Goh, K. I., et al. (2009). An empirical framework for binary interactome mapping.  Nat. Methods  6:83-90.