*2.2. Application Examples with Epitope Peptide-Antibody Immune Complexes*

The ITEM-TWO procedure was tested with immune complexes which were generated by mixing an epitope peptide-containing solution with a solution that contained its respective monoclonal antibody. The mixture of antiFLAG antibody with seven peptides was investigated. The mass spectrum of this antibody-peptide mixture showed in the high mass ranges three narrowly spaced multiply charged ion triplets at each charge state (Figure 6). The molecular masses of these triplet ions (charge states from 21+ to 27+; mean charge state 24.6+) were determined to be 148,730 ± 92 Da, 149,799 ± 45 Da, and 150,785 ± 61 Da, which represented the antiFLAG antibody, the antiFLAG antibody with one bound FLAG peptide, and the antiFLAG antibody with two bound FLAG peptides, respectively (Supplemental Table S5). The mass differences between each ion signal triplet provided rather inaccurate mass values for the bound peptide and, therefore, unambiguous identification of the epitope peptide out of the mixture of seven peptides was not possible.

Due to the chosen quadrupole settings, the low mass ions of unbound peptides were filtered out. Yet, upon raising the collision cell voltage difference there appeared isotopically resolved ions in the low *m*/*z* range of the mass spectrum for the FLAG peptide (see inset in Figure 6) which were recorded with high mass accuracy (20 ppm) and enabled unambiguous identification. It is worth noting that the antiFLAG antibody complex only released the FLAG peptide despite the presence of six other peptides in solution.

By increasing the collision cell voltage differences from 4 V to 200 V in a stepwise manner (20–30 V/step), we observed appearance and incremental rise of doubly and triply charged ion signals in the lower *m*/*z* range together with gradual disappearance of complex ion signals (Figure 6). These relative complex ion intensities, i.e., the heights of apexes of the Gaussian fits, served as the amounts of the various multiply charged ion series of the antibody-peptide complex ions. The height of the apexes of the Gaussian fits of complex-released epitope peptide ion series were used to represent amounts of the released epitope peptides (Supplemental Tables S5 and S6).

∆

() ∆

∆

#

loss from "neutral and resting" RNAse S in the gas phase.

∆

#

 #

∆ **Figure 6.** Nano-ESI mass spectra of FLAG-peptide-antiFLAG antibody complex dissociation. Different collision cell voltage differences (∆**CV**) are shown. (**A**) 20 V. (**B**) 70 V. (**C**) 120 V. (**D**) 150 V. Charge states and *m*/*z* values for selected ion signals are given for the immune complexes (antibody plus one FLAG-peptide and antibody plus two FLAG-peptides; filled orange triangles and filled red diamonds, respectively; right ion series), antiFLAG antibody (open blue circles; right ion series), and FLAG-peptide (open green squares; left ion series). The inset shows a zoom of the singly-charged FLAG peptide ion signals. Solvent: 200 mM ammonium acetate, pH 7.

Again, plotting **norm** (**educts**) vs. ∆**CV**, a sigmoidal shaped course was obtained, which represents the dependence of educt intensities on ∆**CV** (Supplemental Figure S5). Next, the x-axis values (∆**CV**) within the intervals dx above and below ∆**CV**<sup>50</sup> were used to determine the corresponding y-axis values using the equation of the tangent line. The resulting y-axis values, i.e., **norm** (**educts**), enabled the calculation of **ln k** # **mg** values. Plotting increments of **ln k** # **mg** vs. 1 **Tcoll** allowed determination of the part of the apparent dissociation reaction within the "energy regime" located around ∆**CV**<sup>50</sup> (Arrhenius plot; Supplemental Figure S6). In the same manner as was shown above, the calculated value for **k** # **m**0**g** represented the apparent rate constant of dissociation of "neutral and resting" antibody-epitope peptide complexes. Then, **K**# **D m**0**g** and ∆**G**# **m**0**g** (Table 1) were calculated by applying the respective equations.

At last, we performed ITEM-TWO experiments with an epitope peptide that was derived from human cardiac Troponin I (Tn I) against which was directed a monoclonal antiTroponin I antibody (antiTn I). Generation of the immune complex in solution and subsequent electrospraying of the entire mixture started data acquisition with the respective instrument settings as mentioned (for details see Methods section). The mass spectrum of this antibody-peptide mixture showed in the high mass range three narrowly spaced multiply charged ion triplets at each charge state (Figure 7). The molecular masses of these triplet ions (charge states from 23+ to 28+; mean charge state 25.9+) were determined to be 146,414.75 ± 33 Da, 148,218.75 ± 35 Da, and 150,018.88 ± 33 Da, which were identified to be representing antiTroponin I antibody, antiTroponin I antibody with one bound Troponin I epitope peptide, and antiTroponin I antibody with two bound Troponin I epitope peptides, respectively (Supplemental Tables S7 and S8). Again, upon raising the collision cell voltage difference there appeared isotopically resolved ions in the low *m*/*z* range of the mass spectrum for the Troponin I epitope peptide (see insert in Figure 7) which were recorded with high mass accuracy (7 ppm).

— ∆ **Figure 7.** Nano-ESI mass spectra of Tn I-peptide—antiTn I antibody complex dissociation. Different collision cell voltage differences (∆**CV**) are shown. (**A**) 4 V. (**B**) 16 V. (**C**) 30 V. (**D**) 80 V. Charge states and *m*/*z* values for selected ion signals are given for the immune complexes (antibody plus one Tn I-peptide and antibody plus two Tn I-peptides; filled orange triangles and filled red diamonds, respectively; right ion series), antiTn I antibody (open blue circles; right ion series), and Tn I-peptide (open green squares; left ion series). Insert shows a zoom of the triply-charged Troponin I epitope peptide ion signal. Solvent: 200 mM ammonium acetate, pH 7.

 () ∆ ∆ () # # the apparent dissociation reaction within the "energy regime" located around − # # # # binding strengths of "neutral and resting" antibody Again, plotting **norm** (**educts**) vs. ∆**CV**produced a sigmoidal shaped course which represented the dependence of educt intensity on ∆**CV** following Boltzmann characteristics (Supplemental Figure S7). The y-axis values on the tangent line, i.e., **norm** (**educts**), enabled to calculate **ln k** # **mg** values. Plotting increments of **ln k** # **mg** vs. 1 **Tcoll** allowed determination of the part of the apparent dissociation reaction within the "energy regime" located around ∆**CV**50. This Arrhenius plot again provided **ln***A* (pre-exponential factor) as the intercept with the y-axis and − ∆**G**# **mg R** as the slope of the line (Supplemental Figure S8). In the same manner as described above, the value for **k** # **<sup>m</sup>**0**<sup>g</sup>** was calculated. Similarly, by applying the Eyring-Polanyi equation **K**# **D m**0**<sup>g</sup>** was determined. Finally, by using the van´t Hoff equation, ∆**G**# **<sup>m</sup>**0**<sup>g</sup>** was calculated (Table 1) representing binding strengths of "neutral and resting" antibody-epitope peptide complexes.
