**5. Conclusions**

ITEM-TWO is able (i) to determine epitopes and (ii) to investigate the epitopes´ binding strengths in the gas phase. Mixing of antigen or epitope peptide and antibody solutions is the only required in-solution handling step when the complex components are dissolved in electrospray-compatible solutions. From normalized ion intensities, the apparent gas phase quasi equilibrium dissociation constants (**K**# **D m**0**g** ) can be deduced from which apparent dissociation activation energies for neutral and resting immune complexes in the gas phase (∆**G**# **mOg**) can be calculated. As suitable electrospray mass spectrometry equipment has become amply available, our ITEM-TWO method should be easily adaptable by mass spectrometry laboratories all around the world.

**Supplementary Materials:** The following are available online, Equations and theoretical explanations. Figure S1: Course of normalized ion intensities of holo-myoglobin ions (*norm* (*educts*)) as a function of collision cell voltage differences (∆CV), Figure S2: Arrhenius plot for the course of myoglobin dissociation in the gas phase, Figure S3: Course of normalized ion intensities of RNAse S ions (*norm* (*educts*)) as a function of collision cell voltage differences (∆CV), Figure S4: Arrhenius plot for the course of RNAse S dissociation in the gas phase, Figure S5: Course of normalized ion intensities of FLAG-peptide—antiFLAG antibody ions (*norm* (*educts*)) as a function of collision cell voltage differences (∆CV), Figure S6: Arrhenius plot for the course of FLAG-peptide—antiFLAG antibody complex dissociation in the gas phase, Figure S7: Course of normalized ion intensities of Troponin I peptide—antiTroponin I antibody complex (*norm* (*educts*)) as a function of collision cell voltage differences (∆CV), Figure S8: Arrhenius plot for the course of Troponin I peptide—antiTroponin I antibody complex dissociation in the gas phase, Table S1: Ion intensities, charge states, and m/z values for myoglobin at various collision cell voltage difference settings, Table S2: Apex heights and mean charge states of educt and product ion signals upon gas phase dissociation of myoglobin, Table S3: Ion intensities, charge states, and m/z values for RNAse S at various collision cell voltage difference settings, Table S4: Apex heights and mean charge states of educt and product ion signals upon gas phase dissociation of RNAse S, Table S5: Ion intensities, charge states, and m/z values for FLAG-peptide—antiFLAG antibody complex at various collision cell voltage difference settings, Table S6: Apex heights and mean charge states of educt and product ion signals upon gas phase dissociation of FLAG-peptide—antiFLAG antibody complex, Table S7: Ion intensities, charge states, and m/z values for TroponinI peptide—antiTroponinI at various collision cell voltage difference settings, Table S8: Apex heights and mean charge states of educt and product ion signals upon gas phase dissociation of TroponinI immune complex.

**Author Contributions:** Conceptualization: B.D.D. and M.O.G.; methodology: B.D.D., K.F.M.O., and M.O.G.; validation: K.F.M.O., C.R., and C.K.; formal analysis: B.D.D., C.R., and C.K.; investigation: B.D.D. and M.O.G.; resources: M.O.G.; data curation: B.D.D.; writing—original draft preparation: B.D.D. and M.O.G.; writing—review and editing: B.D.D., K.F.M.O., C.R., C.K., and M.O.G.; visualization: B.D.D. and M.O.G.; supervision: M.O.G.; funding acquisition: B.D.D., K.F.M.O., and M.O.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors thank the German Academic Exchange Service (DAAD) for providing a postdoctoral fellowship (Re-invitation Programme for Former Scholarship Holders) for KO (91548123) and a doctoral scholarship for BD (No. 91566064). The WATERS Synapt G2S mass spectrometer has been bought through an EU grant [EFRE-UHROM 9] made available to MOG.

**Acknowledgments:** We express our thanks to Michael Kreutzer for providing his expertise on bioinformatics and to Peter Lorenz and Hans-Juergen Thiesen for providing the FLAG peptide.

**Conflicts of Interest:** The authors declare no conflict of interest.
