Method for detecting protein-protein interactions in a cell utilizing particle-forming polypeptide-bait fusion proteins and virus-like particles

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.

Claims

1. A method of detecting a protein-protein interaction in a cell, the method comprising: expressing in the cell a recombinant fusion protein comprising a viral particle-forming polypeptide and a bait polypeptide of interest, wherein the expressed fusion protein forms a protein-protein complex with an endogenously-expressed prey polypeptide within a virus-like particle; isolating the virus-like particles from the cell; and undertaking a mass spectrometry (MS) analysis of the isolated virus-like particles to detect specific bait-prey protein-protein interactions as compared to background protein-protein interactions from a reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest; thereby detecting the protein-protein interaction in the cell; wherein the viral particle-forming polypeptide is an adenovirus particle-forming polypeptide, a HIV particle-forming polypeptide, or a HCV particle-forming polypeptide.

2. The method according to claim 1, wherein the fusion protein comprises a linker between the viral particle-forming polypeptide and the bait polypeptide of interest.

3. The method according to claim 2, wherein the viral particle-forming polypeptide comprises one or more modifications relative to the naturally occurring viral particle-forming protein, wherein the modification is a deletion, mutation, or functional fragment not inhibiting viral particle formation, or is a fusion protein.

4. The method according to claim 1, wherein the endogenously-expressed prey polypeptide is untagged.

5. The method according to claim 1, wherein the recombinant fusion protein and/or the virus-like particle comprises a purification tag.

6. The method according to claim 5, wherein isolating virus-like particles from the cell occurs via the purification tag prior to detecting the presence of the prey polypeptide.

7. The method according to claim 1, wherein the protein-protein interaction was previously unknown or was previously uncharacterized.

8. The method according to claim 1, further comprising forming virus-like particles comprising the protein-protein complex prior to isolating the virus-like particles from the cell.

9. The method according to claim 1, wherein the reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest comprises a reference set of virus-like particles comprising viral particle-forming polypeptides to a bait polypeptide different from the bait polypeptide of interest.

10. A method of detecting protein-protein interactions in a cell, the method comprising: expressing in the cell recombinant fusion proteins comprising a viral particle-forming polypeptide and a bait polypeptide of interest, wherein the expressed fusion proteins form a plurality of different protein-protein complexes with a plurality of different endogenously-expressed prey polypeptides within virus-like particles; isolating the virus-like particles from the cell; and undertaking a mass spectrometry (MS) analysis of the isolated virus-like particles to detect specific bait-prey protein-protein binding interactions as compared to background protein-protein interactions from a reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest; thereby detecting the protein-protein interactions in the cell; wherein the viral particle-forming polypeptide is an adenovirus particle-forming polypeptide, a HIV particle-forming polypeptide, or a HCV particle-forming polypeptide.

11. The method according to claim 1, wherein the reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest comprises a reference set of virus-like particles comprising viral particle-forming polypeptides to a bait polypeptide different from the bait polypeptide of interest.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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 FKBP12-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.

(2) 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

(3) 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.

(4) 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.

(5) 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.

(6) 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.

(7) 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

(8) 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.

(9) 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).

(10) 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).

(11) Cell Culture, Production and Purification of Virotrap Particles.

(12) HEK293T cells were cultured in a humidified atmosphere at 8% CO.sub.2 using high-glucose DMEM (Invitrogen) complemented with 10% FCS and antibiotics.

(13) 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.sup.6 cells seeded the day before in 75 cm.sup.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.

(14) 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'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.

(15) 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.

(16) For mass spectrometry, 4.75×10.sup.6 HEK293T cells seeded in 75 cm.sup.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 NAPS 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

(17) 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.

(18) 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-GATEWAY® construct (Lievens et al., unpublished) leading to pMET7-Etag-CDK2.

(19) 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).

(20) 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.

(21) TABLE-US-00001 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 FKBP 12  5 GAAACCATCTC amplification Oligo6 CGCTTCTAGATTACTCGAGTGCGGCCGCG FKBP 12  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

(22) 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.sub.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.sub.2 Water was added to a total volume of 150 μl.

(23) 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'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

(24) 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).

(25) 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

(26) 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.

(27) 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

(28) 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 HIV1 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.

(29) 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.

(30) 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.

(31) 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

(32) For the detection of novel interaction partners, the purification procedure was scaled up to compensate for a larger production scale.

(33) 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).

(34) 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).

(35) TABLE-US-00002 TABLE 2 Overview of the proteomics data obtained for different GAG-bait constructs. PRIDE # Unique Experiment # Peptide # Proteins # Proteins proteins FDR Accession # Spectra sequences (all) (>1 peptide) (>1 peptide) (%) Mock 28956 7529 1093 297 158 6 0.4 GAG- 28955 8142 1253 368 193 17 0.4 EGFP GAG- 28954 7618 1237 360 195 16 0.3 CDK2 GAG- 28959 6752 1889 463 277 73 0.7 FADD GAG- 28958 5461 1411 329 173 11 0.7 TRAF3 GAG- 28953 7486 1246 374 218 33 0.4 CKS1B GAG- 28951 7728 1264 260 140 9 0.4 LCP2 GAG- 28952 6768 1197 340 189 7 0.4 GRAP2 GAG- 28957 6399 1361 311 184 21 0.3 S100A 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 (>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.

(36) TABLE-US-00003 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- 8 like 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 F1MSD2 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 LOC525863 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 L18a 2 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 O75131 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

(37) TABLE-US-00004 TABLE 4 List of FADD interaction partners. 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 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.

(38) TABLE-US-00005 TABLE 5 Overview of the proteomics data obtained for different GAG-bait constructs using specific elution of particles from the purification beads. PRIDE # Unique Experiment # Peptide # Proteins # Proteins proteins FDR Accession # Spectra sequences (all) (>1 peptide) (>1 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

(39) 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).

(40) 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.

(41) TABLE-US-00006 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

(42) TABLE-US-00007 TABLE 7 the identified peptides for Desmoglein 1 (DSG1, Swissprot Accession 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

(43) 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.