title: Probing the solution structure of tumor necrosis factor-α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry.
abstract: There are a number of proteins whose active forms are non-covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild-type (wt) proteins. We have used tumor necrosis factor-α (TNF-α) to establish that it is possible to use mass spectrometry to monitor the non-covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5 Da errors mass accuracy of theoretical mass at 50 kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF-α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti-TNF-α antibody. Dissociation and re-association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non-covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non-covalent drug complexes for the purpose of altering biological activity.
authors.affiliation: Centocor Research and Development, a division of J&J PRD, Radnor, PA 19087, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Beil
authors.forename: George A
authors.initials: GA
authors.lastname: Heavner
authors.forename: Sheng-Jiun
authors.initials: SJ
authors.lastname: Wu
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Nemeth
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation; D055503:Protein Multimerization; D058849:Protein Refolding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics
chemical_list: D012997:Solvents; D014409:Tumor Necrosis Factor-alpha; D004121:Dimethyl Sulfoxide