Intact Transition Epitope Mapping- Thermodynamic Weak-force Order (ITEM-TWO)

Most important characteristics of antibodies are that they typically strongly bind to specific epitopes, thereby expressing low dissociation constants (KDs) and high Gibbs free binding energies (delta Gs). In this study, we describe an off-line nano-electrospray mass spectrometry method, termed ITEM-TWO, which enables one to simultaneously identify epitopes and obtain characteristic gas phase dissociation constants of antibody - epitope interactions in a single experiment. To validate the method, we characterize the interaction between an antiHistag antibody and its epitope peptide obtained from a tryptic digest of a His-tag containing beta actin. In a mixture of solution 1 (tryptic digest of a His-tag containing recombinant human beta-actin protein in 30 mM ammonium bicarbonate) and solution 2 (antiHis-tag antibody in 200 mM ammonium acetate) the specific immune complexes form (molar ratio 2.2 : 1). Without any purification steps this mixture (solution 3) is then electrosprayed. With the aid of a quadrupole ion filter, the immune complex ions are separated from unbound peptide ions. Increasing the voltage difference (DeltaCV) in the subsequent collision cell results in collision induced dissociation (CID) by which the epitope peptide(s) is(are) released from the immune complex. The mass(es) of the complex-released peptide(s) is(are) then measured in a ToF analyzer by which the epitope is identified. A step-wise increase in DeltaCV allows the simultaneous determination of the intensities of (i) the surviving ionized immune complexes together with (ii) released epitope peptide ions plus (iii) the left behind antibody ions. From the ions´ normalized intensity ratios are deduced the apparent gas phase dissociation constants (KD#m0g) and the apparent dissociation energies over temperature values (delta G#m0g / T) of the gas phase dissociation processes.