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Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

2019; Elsevier BV; Volume: 18; Issue: 8 Linguagem: Inglês

10.1074/mcp.ra119.001429

ISSN

1535-9484

Autores

Bright D. Danquah, Claudia Röwer, Kwabena F.M. Opuni, Reham F. El‐Kased, David Frommholz, Harald Illges, Cornelia Koy, Michael O. Glocker,

Tópico(s)

Electrohydrodynamics and Fluid Dynamics

Resumo

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody′s epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown. Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody′s epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown. The identification of epitopes or antigenic determinants is essential for the design of novel antibody-based therapeutics and vaccines (1Baraniak I. Kropff B. McLean G.R. Pichon S. Piras-Douce F. Milne R.S.B. Smith C. Mach M. Griffiths P.D. Reeves M.B. 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Mass spectrometric epitope mapping.Mass Spectrom. Rev. 2018; 37: 229-241Crossref PubMed Scopus (59) Google Scholar). Based on our gas phase epitope mapping strategy, termed ITEM-ONE (6Yefremova Y. Opuni K.F.M. Danquah B.D. Thiesen H.J. Glocker M.O. Intact transition epitope mapping (ITEM).J. Am. Soc. Mass. Spectrom. 2017; 28: 1612-1622Crossref PubMed Scopus (15) Google Scholar), where epitopes of known antigens have been identified by precisely determining the mass of the extracted epitope peptide, we have now advanced to ITEM-THREE, where mass spectrometric amino acid sequencing of unknown epitope peptides is performed to identify an antigenic determinant on an antigen surface. Mouse antiRA33 antibody (monoclonal anti-hnRNP-A2/B1; clone DP3B3 lot: 044K4766) was obtained from Sigma-Aldrich (Steinheim, Germany). Rabbit antiTRIM21 antibody (polyclonal anti-52kDa Ro/SSA antibody; sc-20960 lot: F0503) raised against amino acids 141–280 of TRIM21 (52kDa Ro/SSA) of human origin was obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Mouse antiHis-tag antibody (monoclonal antibody MCA 1396; Batch no. 0309) was supplied by Bio-Rad, (Munich, Germany) and mouse antiTNFα antibody (monoclonal antibody; catalogue no. MA5-23720) was produced by ThermoFisher Scientific GmbH (Ulm, Germany). Recombinant human TNF alpha (rhTNFα) was a gift from Prof. Harald Illges, Hochschule Bonn-Rhein-Sieg University of Applied Sciences, Germany. Actin, cytoplasmic 1 recombinant protein was purchased from GenWay Biotech (Catalogue no. 10-288-23014F, San Diego, CA). RA33 peptide (MAARPHSIDGRVVEP-NH2), GPI peptide (ALKPYSPGGPR), Angiotensin II (DRVYIHPF), TRIM21A peptide (LQELEKDEREQLRILGE), TRIM21B peptide (LQPLEKDEREQLRILGE) and TRIM21C peptide (LQELEKDEPEQLRILGE) were synthesized by Peptides and Elephants GmbH (Potsdam, Germany). The synthetic FLAG peptide (DYKDDDDK; article no. 020015) was obtained from ThermoFisher Scientific GmbH and sequencing grade, modified trypsin was obtained from Promega Corporation (Madison, WI). A mixture of equimolar concentrations of seven synthetic peptides (10 μm each; GPI peptide, FLAG peptide, Angiotensin II, TRIM21A peptide, TRIM21B peptide, TRIM21C peptide, and RA33 peptide) was prepared by dissolving the appropriate amounts of the individual lyophilized peptide powders in freshly prepared 200 mm ammonium acetate, pH 7.1 and mixing the appropriate volumes. The peptide mixture-containing solution was shock-frozen and kept at −20 °C until either mass spectrometric analysis or immune complex formation were performed. Recombinant human beta actin (rhβactin) was digested with trypsin (26Glocker M.O. Nock S. Sprinzl M. Przybylski M. Characterization of surface topology and binding area in complexes of the elongation factor proteins EF-Ts and EF-Tu center dot GDP from Thermus thermophilus: A study by protein chemical modification and mass spectrometry.Chemistry. 1998; 4: 707-715Crossref Scopus (30) Google Scholar, 34El-Kased R. Koy C. Lorenz P. Montgomery H. Tanaka K. Thiesen H. Glocker M. A novel Mass spectrometric epitope mapping approach without immobilization of the antibody.J. Proteomics Bioinform. 2011; 4 (001–009)Google Scholar, 45Glocker M.O. Arbogast B. Deinzer M.L. Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests.J. Am. Soc. Mass Spectrom. 1995; 6: 638-643Crossref PubMed Scopus (28) Google Scholar) using a modified Filter Aided Sample Preparation (FASP) protocol. To 10 μl of 200 mm DTT, dissolved in 0.1 m Tris/HCl containing 8 m urea was added 20 μl of rhβactin solution (protein concentration 1 μg/μl). This mixture was incubated at 37 °C for 30 min. Then, this solution was transferred into an equilibrated 30K Amicon centrifugal filter (equilibration with 1% formic acid according to protocol (46Yang J. Rower C. Koy C. Russ M. Ruger C.P. Zimmermann R. von Fritschen U. Bredell M. Finke J.C. Glocker M.O. Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.Methods. 2015; 89: 30-37Crossref PubMed Scopus (6) Google Scholar)) and 170 μl of 8 m urea in 0.1 m Tris/HCl, pH 8.5, were added and centrifuged at 13,000 rpm for 15 min. After discarding the filtrate, 150 μl of 8 m urea in 0.1 m Tris/HCl, pH 8.5, were added to the retentate in the filter unit and centrifuged again at 13,000 rpm for 15 min. A further wash of the retentate was done by adding 100 μl of 8 m urea in 0.1 m Tris/HCl, pH 8.5, to the filter unit and centrifuging at 13,000 rpm for 12 min. The filtrates were discarded and the retentate was further washed for three times, first by adding 100 μl, then 75 μl, and lastly 50 μl of 50 mm ammonium bicarbonate solution, pH 8.6, and each time centrifugation was performed at 13,000 rpm for 10 min, 12,000 rpm for 8 min, and 12,000 rpm for 6 min, respectively. After the three washings, the filter unit containing the retentate (ca. 5 μl) was transferred into a new collection tube. A volume of 35 μl of 11.42 ng/μl of trypsin in 50 mm ammonium bicarbonate, pH 8.6, was added to the protein that was dissolved in the solution on the filter unit to obtain an enzyme/substrate ratio of 1:50 (w/w). The mixture was incubated at room temperature in a wet chamber for 16 h and then centrifuged at 8000 rpm for 5 min and at 12,000 rpm for 3 min. Next, a volume of 40 μl of 10 mm ammonium bicarbonate, pH 8.6, and a further amount of 4 μl of 0.1 μg/μl trypsin solution (composition see above) was added and incubated at 37 °C for 2 h. Finally, the mixture was centrifuged at 12,000 rpm for 8 min and the filtrate (ca. 80 μl), which contained the tryptic peptides, was collected for further analysis. The peptide concentration of the solution was determined using the Qubit® 2.0 Fluorometer (Carlsbad, CA), following described procedures (6Yefremova Y. Opuni K.F.M. Danquah B.D. Thiesen H.J. Glocker M.O. Intact transition epitope mapping (ITEM).J. Am. Soc. Mass. Spectrom. 2017; 28: 1612-1622Crossref PubMed Scopus (15) Google Scholar, 47Yefremova Y. Melder F.T.I. Danquah B.D. Opuni K.F.M. Koy C. Ehrens A. Frommholz D. Illges H. Koelbel K. Sobott F. Glocker M.O. Apparent activation energies of protein-protein complex dissociation in the gas-phase determined by electrospray mass spectrometry.Anal. Bioanal. Chem. 2017; 409: 6549-6558Crossref PubMed Scopus (11) Google Scholar). Aliquots (10 μl, each), were shock-frozen and kept at −20 °C until either mass spectrometric analysis or immune complex formation were performed. Tryptic digestion of recombinant human TNF alpha (26Glocker M.O. Nock S. Sprinzl M. Przybylski M. Characterization of surface topology and binding area in complexes of the elongation factor proteins EF-Ts and EF-Tu center dot GDP from Thermus thermophilus: A study by protein chemical modification and mass spectrometry.Chemistry. 1998; 4: 707-715Crossref Scopus (30) Google Scholar, 34El-Kased R. Koy C. Lorenz P. Montgomery H. Tanaka K. Thiesen H. Glocker M. A novel Mass spectrometric epitope mapping approach without immobilization of the antibody.J. Proteomics Bioinform. 2011; 4 (001–009)Google Scholar, 45Glocker M.O. Arbogast B. Deinzer M.L. Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests.J. Am. Soc. Mass Spectrom. 1995; 6: 638-643Crossref PubMed Scopus (28) Google Scholar) (rhTNFα, 1 μg/μl) was performed by adding 15 μl of the rhTNFα dissolved in 200 mm ammonium acetate, pH 7.1, to 32 μl of trypsin solution (9.4 ng/μl in 4.8 mm Tris/HCl with 5 mm DTT) to yield an enzyme/substrate ratio of 1:50 (w/w). This mixture was incubated at 37 °C for 20 h. The resulting tryptic peptide solution was divided into nine aliquots, each of which contained a volume of 5 μl. Each aliquot was desalted by loading the entire 5 μl volume onto one C18 ZipTip Pipette Tip (Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co. Cork, Ireland) that had been wetted with a mixture of deionized H2O/ACN (50:50, v/v). Equilibration and washing solutions consisted of 1% HCOOH in deionized H2O; two times 10 μl were used for each step. The affinity-bound peptides were eluted into 5 μl of 1% HCOOH in H2O : 1% HCOOH in ACN (50:50, v/v) (46Yang J. Rower C. Koy C. Russ M. Ruger C.P. Zimmermann R. von Fritschen U. 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