Patent Application: US-201314402667-A

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:
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 . 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 ). 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 ). hek293t cells were cultured in a humidified atmosphere at 8 % co 2 using high - glucose dmem ( invitrogen ) complemented with 10 % fcs and antibiotics . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 ). 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 ( fig2 ). 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 . 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 ( fig3 ). 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 . 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 . 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 . fig4 , 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 ( fig4 , 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 . 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 . 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 ( fig4 , panel d ). as controls , irrelevant bait and prey constructs were used . 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 . fig5 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 . for the detection of novel interaction partners , the purification procedure was scaled up to compensate for a larger production scale . 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 ( fig6 , 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 ( fig6 , 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 ( fig6 , 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 ) ( fig6 , panel b , right side table ). 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 ( fig6 , panel c ). 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 . 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 ). fig7 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 . 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 - 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