METHOD FOR IDENTIFICATION OF VIRUSES AND DIAGNOSTIC KIT USING THE SAME
20240011989 ยท 2024-01-11
Inventors
Cpc classification
International classification
G01N33/543
PHYSICS
Abstract
This invention describes a novel method for identification of known and unknown viruses from various biological and non biological samples using carrier suitable for immobilisation of membrane proteins of different target cells of human, animal or bacterial origin for capturing the virus, whose peptides are identified by mass spectrometry analysis, a diagnostic kit based on the said method and the use thereof.
Claims
1. A method for identification of viruses in a sample comprising following steps: a) collecting and optionally concentrating the sample; b) isolation of membrane proteins of target cells; c) coupling of the membrane proteins isolated in step b) to a carrier suitable for immobilisation of proteins obtaining carrier with immobilised membrane proteins; d) incubating sample with preparation obtained in step c); e) separating carrier with immobilised membrane proteins having attached the virus particle; f) detaching the virus particle from the carrier with immobilised membrane proteins; g) preparation of the sample for the MS (mass spectrometry) analysis; h) determination of the viral peptide sequence/s obtained by step g); and i) identification of the virus by comparing the peptide sequence/s identified in step h) with databases of known virus peptide sequences (known virus) or, using sequences identified in step h) for identification of a new virus by the RNA/DNA analysis methods (unknown virus).
2. The method according to claim 1, wherein the sample is a biological sample derived from blood, body liquids such as liquor, saliva, any tissue sample of a human or animal origin or any swab taken from human or animal subject.
3. The method according to claim 1, wherein the sample is non-biological sample, preferably environmental sample taken from river, lake, sea, water conduit, water works, ventilation system and like, sample taken from soil or swab taken from any object either in the human or animal living space, sample from any industrial process such as food processing, biotechnological and pharmaceutical process.
4. The method according to claim 1, wherein target cells are selected from the group comprising: primary cells, any immortalised or tumour cell lines from different origin in human or animal body; or any bacterial strain cells.
5. The method according to claim 1, wherein carrier suitable for immobilisation of proteins is selected from the group comprising: magnetic beads, agarose beads and like.
6. Use of method according to claim 1, for identification of known viruses or unknown viruses.
7. Carrier suitable for immobilisation of proteins linked to the membrane proteins of target cells, obtained by step c) of claim 1.
8. Carrier suitable for immobilisation of proteins linked to the membrane proteins of target cells according to claim 7, wherein target ceils are selected from the group comprising: primary cells, any immortalised or tumour cell lines from different origin in human or animal body; or any bacterial strain cells.
9. Carrier suitable for immobilisation of proteins linked to the membrane proteins of target cells according to claim 7 for use in identification of known viruses or unknown viruses.
10. A diagnostic kit for identification of viruses using the method according to claim 1 comprising: carrier suitable for immobilisation of the membrane proteins of target cells obtained by step c) of claim 1, leaflet containing instructions for use and optionally chemicals needed for carrying out steps e)-g) of claim 1.
11. The diagnostic kit according to claim 10, wherein target cells are selected from the group comprising: primary cells, any immortalised or tumour cell lines from different origin in human or animal body; or any bacterial strain cells.
12. Use of the diagnostic kit according to claim 10, for identification of known viruses or unknown viruses.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0012]
[0013]
[0014]
[0015]
DETAILED DESCRIPTION OF THE INVENTION
[0016] This invention relates to the method for identification of known and unknown viruses in a sample, which does not require a priori knowledge of potential pathogens. Method according to this invention comprises following steps: [0017] a) collecting and optionally concentrating the sample [0018] b) isolation of membrane proteins of target cells [0019] c) coupling of the membrane proteins isolated in step b) to a carrier suitable for immobilisation of proteins obtaining carrier with immobilised membrane proteins [0020] d) incubating sample with preparation obtained in step c) [0021] e) separating carrier with immobilised membrane proteins having attached the virus particle [0022] f) detaching the virus particle from the carrier with immobilised membrane proteins [0023] g) preparation of the sample for the MS (mass spectrometry) analysis [0024] h) determination of the viral peptide sequence/s obtained by step g) [0025] i) identification of the virus by comparing the structure/s identified in step h) with databases of known virus peptide sequences (known virus) or, using sequences identified in step h) for identification of a new virus by the RNA/DNA analysis methods (unknown virus).
[0026] In one embodiment of the invention sample to be tested is a biological sample derived from blood, body liquids such as liquor, saliva and the like and any tissue sample of a human or animal origin or any swab taken from human or animal subject.
[0027] In another embodiment of the current invention sample is non-biological sample, preferably environmental sample taken from river, lake, sea, water conduit, water works, ventilation system and like, sample taken from soil or swab taken from any object, preferably object present in the human or animal living space, industry process quality control where the said industry is food processing industry, pharmaceutical or biotechnology based industry.
[0028] The sample to be tested could be used directly in the form taken or optionally prepared for testing by concentrating used known methods (e.g. from water: Cashdollar J L and Wymer L.: Methods for primary concentration of viruses from water samples: a review and meta-analysis of recent studies. J Appl Microbiol., 115(1):1-11, 2013, doi: 10.1111/jam.12143).
[0029] Target cells are selected from the group comprising: primary cells, any immortalised or tumour cell lines from different origin in human or animal body; or any bacterial strain cells.
[0030] The term primary cells means that it is a population of cells from a multicellular organism that are taken directly from living tissue (e.g. biopsy material) and established for growth in vitro. These cells have undergone very few population doublings.
[0031] The term immortalized cells means that it is a population of cells from a multicellular organism due to mutation, escape normal cellular senescence and keep undergoing division. Thus, this kind of cells can grow in vitro for prolonged periods
[0032] The term cancer cells means that this is a population of cells from a multicellular organism that divide relentlessly, forming solid tumors or flooding the blood with abnormal cells.
[0033] Isolation of membrane proteins of target cells are performed using method standard in the art (Smith S M, Strategies for the Purification of Membrane Proteins in Walls D and Loughran S T (eds.) Protein Chromatography, Methods and Protocols, Methods in Molecular Biology, 2011, 681:485-496, doi: 10.1007/978-1-60761-913-0_29, Springer Science+Business Media; Lai X, A Reproducible Method to enrich Membrane Proteins with High-purity and High-yield for an LC-MS/MS approach in quantitative membrane proteomics. Electrophoresis, 2013, 34(6):809-817, doi: 10.1002/elps.201200503). Carrier suitable for immobilisation of proteins according to this invention is described as any carrier suitable for immobilisation of proteins and it is preferably selected from the group comprising: magnetic beads, agarose beads and the like (Meldal M and Schoffelen S, Recent advances in covalent, site specific protein immobilization. F1000Research 2016, 5:2303; Zucca P et al. Agarose and Its Derivatives as Supports for Enzyme Immobilisation. Molecules 2016, 21:1577, doi: 10.3390/molecules21111577; Mohamad N R et al. An Overview of Technologies for Immobilisation of Enzymes and Surface Analysis Techniques for Immobilized Enzymes, Biotechnology & Biotechnological Equipment, 2015, 29(2), 205-220, doi: 10.1080/13102818.2015.1008192).
[0034] Therefore, the different embodiments of this invention could be achieved by changing the type of target cells and consequently the receptors on their surface, and further coupling of membrane proteins from different targets cells to carrier suitable for immobilisation of proteins which are consequently then capable to attach viruses having specificity for exact receptor exposed on the surface of the particular cell, wide variety of human, animal or bacterial viruses can be detected and identified.
[0035] Viruses are detached from the carrier with immobilised membrane proteins by method know in the art (Protein Purification Protocols, doi: 10.1385/159259655X) and prepared for the MS analysis according to standard methodology known in the art (Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow, doi: 10.1002/0471142727.mb1025s88). Peptide sequences are then analysed and their structure compared to databases of known proteins (for example Swiss or Uniprot).
[0036] In one embodiment the method according to the invention is used for identification of known viruses in case of finding the peptide structure which resulted from MS analysis in the databases of known viruses. Presence of viral DNA or RNA can be verified by standard PCR or RT-PCR methodology (Ambriovid Ristov A. et al. (Ed) (2007) Metode u molekularnoj biologiji, Institut Ruder Bokovic, Zagreb, ISBN: 978-953-6690-72-5).
[0037] In yet another embodiment the method according to the invention is used for characterisation of unknown viruses. In this peptide structure which resulted from MS analysis could not be found in the databases of known viruses. In this case also Presence of viral DNA or RNA can be verified by standard PCR or RT-PCR methodology.
[0038] Present invention further encompasses carrier with immobilised membrane proteins of target cells for use in identification of known viruses or characterisation of unknown viruses.
[0039] Present invention further describes diagnostic kit comprising carrier with immobilised membrane proteins of target cells, leaflet containing instructions for use and optionally chemicals needed for carrying out steps e)-g) of the method according to the invention.
[0040] Target cells used for developing the diagnostic kit according to the invention could be selected from the group comprising: primary cells, any immortalised or tumour cell lines from different origin in human or animal body; or any bacterial strain cells.
[0041] By varying the target cells used for developing of the said diagnostic kit, different diagnostic sets could be developed which are capable for detecting for example human respiratory viruses wherein respiratory tract cells are used as target cells. Another example may include diagnostic kit based on liver cells for detection of liver pathogen viruses, cells from nervous system to detect viruses capable of infecting brain and spinal cord and cells from skin to detect viruses causing skin infection. These are only given as an example which does not in any case limit the spectrum of the target cells and therefore the diagnostic kit based on thereof.
[0042] Present invention also describes use of the diagnostic kit to detect the known and unknown virus in a sample to be tested which according to the invention could be a biological sample derived from blood, body liquids such as liquor, saliva and the like and any tissue sample of a human or animal origin or any swab taken from human or animal subject.
[0043] In another embodiment of the current invention sample is non-biological sample, preferably environmental sample taken from river, lake, sea, water conduit, water works, ventilation system and like, sample taken from soil or swab taken from any object, preferably object present in the human or animal housing, industry process quality control where the said industry is food processing industry, pharmaceutical or biotechnology based industry.
Examples
Experimental Model
[0044] As experimental model for demonstration of technical viability of this invention and obtaining experimental proofs of the concept in general, pathogen adenovirus type 5 (Ad5) was chosen together with two stably transfected cell clones of human rhabdomyosarcoma cells (RD-A7 and RD-G7) which express primary receptor for Ad5 at the cell surface, namely coxsackievirus and adenovirus receptor (CAR) (Nemerow et al., Virology. 2009 Feb. 20; 384(2):380-8, Majhen et al. Life Sci. 2011 Aug. 15; 89(7-8):241-9). Wild type RD cells have negative CAR phenotype and in the following experiments were used as negative control.
Methodology
[0045] 1. Cultivation of RD, RD-A7 and RD-G7 cells
[0046] The human rhabdomyosarcoma (RD) cell line was obtained from the American Type Culture Collection (CCL-136, ATCC; USA). RD-A7 and RD-G7 cells were obtained as described in Majhen et al. Life Sci. 2011 Aug. 15; 89(7-8):241-9. Cells were grown in vitro in Petri dishes having diameter of 10 cm in the moisture saturated atmosphere at 37 C. with 5% CO.sub.2. Dulbecco's modificated Eagle's cultivation medium with addition of 10% of fetal bovine serum (DMEM-FBS medium; Fetal Bovine Serum, Sigma F7524; Dulbecco's Modified Eagle's Mediumhigh glucose, Sigma D5796) was used for cultivation. After 3 days cells were inoculated in fresh medium in order to avoid dying because of the exhaustion of the cell growth medium.
2. Thawing, Sub-Culturing and Freezing of RD, RD-7 and RD-G7 Cells
[0047] Ampoules with frozen cell were taken out of the liquid nitrogen and incubated in water bath until completely defrosted. Cell were transferred to the Petri dishes having diameter of 10 cm containing 9 mL of the DMEM-FBS medium, warmed up previously to 37 C. in a water bath. Next day, after the check-up under light microscope, the growth medium has been replaced, and according to requirements, cells were sub-cultured.
[0048] When sub-culturing, DMEM-FBS was removed from Petri dishes and the cells were washed with previously warmed up trypsin, 0.25%, Trypsin-EDTA solution, Sigma T4049 37 C.). After washing, cells were incubated in 1 mL of fresh trypsin until they start to detach from the bottom. Trypsin action was blocked by adding 9 ml of the DMEM-FBS medium (previously warmed up to 37 C. in a water bath). Cell were then resuspended by multiple pipetting up and down and evenly distributed to new Petri dishes so as to have 1.510.sup.6 cells per each.
[0049] For the purpose of freezing, cells were detached from the bottom with the trypsin solution as described above. After counting of cells, cell suspension was transferred to 15 ml plastic tube and centrifuged at 1100g during 10 min. Cell pellet was resuspended in the 950 L of the DMEM-FBS medium, and cryopreservation agent DMSO at final concentration of 5% was added to the cell suspension. Ampoules for freezing were kept on ice during 30 minutes and then added to the rack of the liquid nitrogen container (80 C.).
3. Mycoplasma Test
[0050] RD, RD-7 and RD-G7 cells (510.sup.3 cell per each cell line) were grown on microscope slides using DMEM-FBS growth medium without antibiotics. Five days from inoculation, cells were fixed with acetic acid:methanol (3:1) and incubated with Hoechst 33258 dye (50 ng/mL in PBS) in dark at room temperature for 10 min. Cells were washed with deH.sub.20, and mounted using mounting solution (22.2 mM citric acid and 55.6 mM Na.sub.2HPO.sub.4 in 50% glycerol, pH 5.5) and inspected by fluorescent microscope.
4. Assessing Expression of CAR in the Cell RD, RD-A7 and RD-G7 Using Flow Cytometry Method
[0051] RD, RD-A7 and RD-G7 cells were trypsinized and centrifuged 10 min at 1100g and at room temperature and the medium in supernatant was discarded. Cell pellet was washed for the first time with 5 mL and second time with 8 mL of PBS which did not contain Ca++ and Mg++. Cell were centrifuged again for 10 min at 1100g and at room temperature and resuspended in 10 mL of cold PBS without Ca++ and Mg++. From each sample, 510.sup.5 cell/50 l were transferred to the flow cytometry tubes. Cells were incubated with primary antibody murine mAb anti-CAR, clone RmcB (Upstate Cell Signaling Solutions, USA) for 1 h on ice with occasional shaking of the tubes in order for antibody to be evenly distributed. After incubation, samples were washed 2 times with 450 L of PBS without Ca++ and Mg++ and secondary antibody FITC Goat Anti-Mouse IgG Clone Polyclonal (RUO), BD Bioscience, was added. Incubation with secondary antibody lasted 30 min on ice with occasional gentle shaking of tubes in order for antibody to be evenly distributed. Cells were washed for three times with 450 L cold PBS without Mg and Ca, and pellet was finally dissolved in 400 L of 0.1% BSA in PBS without Ca++ and Mg++, after which expression of CAR was measured using flow cytometer. Antibodies used in the experiment are shown in Table 1.
TABLE-US-00001 TABLE 1 Final concentration and volume of primary and secondary antibodies used in measurement of CAR expression by flow cytometry method. ANTIBODY CONCENTRATION/g/mL VOLUME/ L Primary IgG1 isotype control 3.63 1.0 Antibodies RmcB, anti-CAR 14.54 0.8 Secondary Goat antibodies 9.09 1.0 Antibodies against mouse immunoglobulins
5. Isolation of Membrane Proteins from RD, RD-A7 and RD-G7 Cell Lines
5.1. Abcam Plasma Protein Extraction Kit (ab65400)
[0052] Cell lines were grown using methodology described in the above section 1 until minimum of 510.sup.8 cells was reached per each cell line. On the cell collection day for the purpose of isolation of plasma membrane proteins, one of the Petri dish of each cell line was used for counting the cells. The total number of cell of the same clone was obtained by multiplying of the cell number in one Petri dish with the number of Petri dishes of the same clone.
[0053] Cells were collected by scratching in cold PBS and then centrifuged for 10 min at 1100g. Pellet was washed with 3 mL of cold PBS and resuspended in 2 mL of homogenisation buffer in cold Dounce homogenizer. Cells were homogenised on ice 50 times. Plasma membrane proteins from RD, RD-A7 and RD-G7 cell lines were isolated by making use of Abcam Plasma Protein Extraction Kit (ab65400) using the manufacturer's instructions and kept at 80 C. in PBS with 0.5% Triton X-100.
5.2. Isolation of Membrane Proteins Using Ultracentrifugation
[0054] Dry pellets of RD, RD-A7 and RD-G7 cells (10.sup.8 cells from each cell line) were washed with 3 mL of PBS without Ca++ and Mg++ by centrifuging at 1100g, 10 min. Pellets were then resuspended in 2 mL of homogenization buffer from Abcam Plasma Protein Extraction Kit (ab65400) with addition of protease inhibitor according to manufacturer's instructions and homogenized in previously cooled Dounce homogenizer with larger pestle (Dounce tissue grinder set, Sigma D8938; pestle A clearance 0.0030-0.0050 in., pestle B clearance 0.0005-0.0025 in., working volume1.2 mL60 mm) 100 times. In order to remove cells that were not broken down together with nuclei, homogenates were centrifuged at 1000g, 5 min at 4 C. Supernatants were transferred to ultracentrifuge cuvettes and centrifuged at 15000g, 20 min at 4 C. In this step mitochondria and larger cell elements are pelleted and supernatants were transferred into new ultracentrifuge cuvettes and centrifuged at 100000g at 4 C. during 1 h. Supernatants were discarded and the pellets containing membrane proteins were dissolved in 100 l PBS without Ca++ and Mg++ with 0.5% Triton X-100. Samples were then sonicated 3 times for 3 seconds, aliquoted and kept at 80 C.
6. Determination of the Concentration of Total Isolated Membrane Proteins with BCA Method
[0055] Concentrations of total isolated membrane proteins from cell lines RD, RD-A7 and RD-G7 were determined by making use of Pierce.sup.TC BCA Protein Assay Kit according to manufacturer's instructions. All samples were diluted 10 and transferred to the plate in duplicates. Absorbance was measured at wavelength 570 nm.
[0056] Calibration curve was made using standard BSA concentrations (125, 250, 500, 1000 and 1500 g/mL) and concentration of proteins to be measured were calculated using formula: c (g/mL)=(Absb) (100*a), where c is protein concentration, Abs final absorbance, b is ordinate segment and a plunge of the axis.
7. Western Blot Analysis of CAR in Isolated Membrane Proteins
[0057] Samples of isolated membrane proteins of RD, RD-A7 and RD-G7 cell lines were incubated in non-reducing buffer (Tris 1M pH 6.8, SDS 10%, glycerol 4 mL, Brophenol Blue 20 mg, mqH.sub.2O 5 mL) during 10 min at 37 C. and then loaded on 12% sodium dodecyl sulfate polyacrylamide gel, 30 g per well. Proteins were subjected to electric current with constant voltage of 80 V during 30 min, 100 V during 1.5 h. Upon electrophoresis, samples were transferred to nitrocellulose membrane under constant current of 400 A during 90 min. The membrane was washed in TBST (Tris buffered saline with Tween-20: 100 mM TrisHCl, 1.5M NaCl, 0.5% Tween-20, pH 7.5) and blocked using PBS with 5% nonfat dry milk and 0.1% Tween-20 in order to prevent nonspecific bonding of the primary antibody. After that membrane was incubated with primary antibody anti-CAR (RmcB clone) which was diluted 1:1000 in TBST with 5% nonfat dry milk overnight at 4 C. Next day, membrane was washed again with TBST and incubated with secondary antibody against mouse immunoglobulins diluted 1:5000 in TBST with 5% nonfat dry milk on a shaker during 2 h at room temperature. Membrane was washed with TBST and incubated with a chemiluminescence reagent during 1 min, exposed to X-ray film which was developed in the dark room.
8. Coupling Membrane Proteins on Dynabeads M-280 and Incubation of Conjugated Magnetic Particle with Adenovirus Type 5
[0058] Membrane proteins isolated from RD, RD-A7 and RD-G7 cell lines were conjugated with Dynabeads M-280 Tosylactivated according to manufacturer's instructions using magnets DynaMag-2. Incubation of magnetic particles with membrane proteins took place on rotor overnight at 5 rpm and at 4 C. Mass of the incubated magnetic particles and membrane proteins are shown in Table 2.
TABLE-US-00002 TABLE 2 Conditions for coupling of magnetic particles with membrane proteins Cell lines from which membrane proteins were Mass (volume) of Mass (volume) of isolated magnetic particles membrane proteins RD 5.00 mg (165.00 L) 100 g (11.25 L) RD-A7 5.00 mg (165.00 L) 64.97 g (10.10 L) RD-G7 5.00 mg (165.00 L) 100 g (7.97 L)
[0059] Magnetic particles with immobilised membrane proteins were incubated with 10 L of Adenovirus type 5 (Ad5) (Majhen et al., Biochem Biophys Res Commun. 2006 Sep. 15; 348(1):278-87) having concentration of 7.1410.sup.11 pp/mL overnight at 5 rpm at 4 C. Magnetic particles were then washed with 0.1% BSA in PBS, pH 7.4, in order to remove non-bound and non-specifically bound Ad5 particles. Elution of the linked Ad5 particles from conjugated magnetic particles was performed by changing of ionic strength with 0.845 M NaCl (100 L) on a shaker during 30 min at 700 rpm and at room temperature.
[0060] Eluates of Ad5 from conjugated magnetic beads were used final analysis using mass spectrometry.
9. LC-MS Non Targeted Mass Spectrometry Analysis
[0061] Analysis was done using following instruments and software: Autoflex Speed MALDI TOF/TOF, Bruker, Germany; Dionex Ultimate 3000 RSLCnano System, Thermo Scientific, SAD; Proteineer fcII, Bruker, Germany; FlexControl 3.4, Bruker, Germany; ProteinScape 3.0, Bruker, Germany; Hystar 3.2, Bruker, Germany; WARP-LC 1.3, Bruker, Germany; Chromeleon Xpress 6.8, Thermo Scientific, SAD. Sample digestion was done by using Trypsin over 18 h at 37 C. with shaking. Peptide separation was done by Dionex Ultimate 3000 RSLC nano System with UV/VIS detector (Thermo Scientific, SAD). Belonging column for peptide purification and separation was used. Peptide fractions were collected and deposited onto MALDI plate by using Proteinees fcII, Bruker, Germany. Peptide analysis was performed by mass spectrometer Autoflex Speed MALDI TOF/TOF (Bruker, Germany). External spectral calibration was done by cubic enhanced algorithm using signals obtained by recording spectra from peptides of known masses. MS and MS/MS spectra identification was done by software ProteinScape 3.0, Bruker, Germany. For verifying identifying peptides from the virus sample human Ad5 (unreviewed) data base from www.uniprot.org was used.
10. LC-MS Targeted Mass Spectrometry Analysis
[0062] Analysis was done using following instruments and software: 6460 Triple Quad LC/MS Agilent technologies, SAD; 1290 Infinity LC System, Agilent Technology, SAD; chromatographic column Acquity UPLC BEH C18 1.7 m, 2.1150 mm, Waters, SAD; MassHunter Workstation software, LC/MS Data acquisition B.07.00, Agilent Technologies, SAD. Purified Ad5 samples used in this analysis were first desalted and then digested by using RapiGest SF Surfactant (Waters, SAD). RD and RD-G7 samples were prepared in same way as for non-targeted analysis. Peptide separation was done by 1290 Infinity LC System directly connected to 6460 Triple Quad LC/MS. MS spectra obtained for Ad5 was used as a reference for identification of peptides obtained in MS of RD and RD-G7 samples.
Results
1. Mycoplasma TestNegative Result
[0063] All three cell lines (RD, RD-A7 and RD-G7) were tested for presence of mycoplasma in order to confirm that samples were not contaminated by this frequent contaminant.
2. Confirmation of RD-A7 and RD-G7 Expression of CAR on their Surface
[0064] In order to check whether human adenovirus type 5 (Ad5) primary receptor (CAR) is present at the surface of RD, RD-A7 and RD-G7 cell lines, flow cytometry method using anti-CAR (RmcB) antibodies were performed. Results show (see
3. Measurement of Concentration of Membrane Proteins Isolated from RD, RD-A7 and RD-G7 Cell Lines
[0065] Table 3. shows concentrations of membrane proteins isolated from RD, RD-A7 and RD-G7 cell lines used in proteomic analysis described in the section 6 of Methodology part.
TABLE-US-00003 Concentrations of membrane proteins isolated from RD, RD-A7 and RD-G7, used in proteomic analysis. Sample c (g/L) m (g) RD 8,882 1332,30 RD-A7 9,817 1472,55 RD-G7 12,542 1881,30
4. Presence of CAR in Isolates of Membrane Proteins from RD-A7 and RD-G7 Cell Lines was Confirmed by Western Blot Analysis
[0066] Fractions of membrane proteins isolated from RD, RD-A7 and RD-G7 cell lines were analyzed by Western blot analysis in order to check whether CAR was present.
5. Adenovirus Type 5 Proteins were Identified with Mass Spectrometry in Eluates of the Magnetic Beads Coupled to the RD-A7 and RD-G7 Membrane Proteins
[0067] Magnetic beads with immobilised membrane proteins isolated from RD, RD-A7 and RD-G7 cells were incubated with 10.sup.10 pp Ad5 according to protocol described in the section 8 of the Methodology part. Eluates of the content bound to the beads with immobilised membrane proteins were sent to the mass spectrometry analysis Following samples were analysed: Ad5 incubated with magnetic beads coupled to membrane proteins isolated from RD cells (negative control), Ad5 incubated with magnetic beads coupled to membrane proteins isolated from RD-A7 cells and Ad5 incubated with magnetic beads coupled to membrane proteins isolated from RD-G7.
[0068] MALDI-TOF/TOF analysis was performed and after search of the SwissProt database it was evident that Ad5 was present in the eluates of the magnetic beads coupled to the the RD-A7 and RD-G7 cell membrane proteins while in the eluates of the beads coupled to the RD cell membrane proteins only BSA and trypsin were detected (present due to the preparation protocol). Identified protein and peptides are shown in table 4 and spectra of identified Ad5 protein is shown in
TABLE-US-00004 TABLE4 Adenovirustype5proteinsidentifiedinthe eluatesfrommagneticbeadscoupledtotheRD, RDAFandRDG7cellImesmembraneproteins Cell ProteinAd5 Accession Noof line identified (SwissProt) peptides Score Sequence RD / / / / / RDAZ Prehistone NPADE02 1 135.37 R.TTVDDAIDAVVEFAR.N= like SEQIDNo.1 nucleoprotein RDG7 Hexon CAP9_ADE02 1 96.36 R.GIVTDFAFLSPLASSAASR.S= interlacing SEQIDNo.2 protein Prehistone NPADEC02 3 237.79 R.APWGAHK.R= like SEQIDNo.3 nucleoprotein R.TTVDDAIDAVVEEAR.N= SEQIDNo.1 R.NYTPTPPPVSTVDAAIQTVVR.G= SEQIDNo.4
[0069] Additionally, targeted LC-MS analysis was done on eluates from magnetic beads coupled to membrane proteins isolated from RD and RD-G7 cell lines. Identification of the detected peptides was done using in house made data base of Ad5. In LC-MS analysis was evident that Ad5 was present in the eluates of the magnetic beads coupled to membrane proteins isolated from RD-G7 cells while in the eluates of the beads coupled to membrane proteins isolated from RD cells there was no peptides detected. Identified proteins are listed in table 5 and representative MRM chromatogram of one of the identified Ad5 protein is shown in
TABLE-US-00005 TABLE5 Adenovirustype5proteinsidentifiedin targetedLCMSanalysisintheeluates frommagneticbeadscoupledtomembrane proteinsisolatedfromRDandRDG7cell lines. Cell ProteinAd5 line identified Sequence Rd / / RDG7 Prehistone VLAIVNALAENR= like SEQIDNo.5 nucleoprotein LSAILEAVVPAR= SEQIDNo.6 LLLLLLAPFTDSGSVSR= SEQIDNo.7