*2.1. Procedure Development with Myoglobin and Application to RNAse S Dissociation*

The mass spectral data obtained from electrospraying myoglobin (the holo-myoglobin complex consists of apo-myoglobin plus heme) were collected by following the ITEM-TWO protocol (see Methods section for in-solution handling and data acquisition steps). The ESI mass spectrum of myoglobin (Figure 4) provides an ion series of multiply charged ions from which a mean charge state of 8.1+ and an average mass of 17,566.95 ± 0.46 Da is calculated for holo-myoglobin. After having electrosprayed the myoglobin solution, and upon having switched on the collision gas and having increased the collision cell voltage difference (∆**CV**) in a step-wise manner (5–20 V/step), dissociation of holo-myoglobin complexes caused appearance of complex-released heme ions (ligands) in the low mass ranges of the mass spectra (Supplemental Table S1). The mass spectra also showed ion signals of apo-myoglobin (mean charge state: 7.2+) in the high mass ranges (average mass: 16,951.46 ± 0.44 Da) with increasing yields (Figure 4). The *m*/*z* value of the ion that appeared in the low mass range ([heme]+) was 616.21 and corresponded precisely to the calculated values for singly protonated [heme]+ (*m*/*z* 616.18), resulting in a mass accuracy of 48 ppm.

Mean charge states of holo-myoglobin ions (m+), apo-myoglobin (n+) and heme (p+) as well as apex heights (holo-myoglobin ions (h, educts), heme ions (i, product), and apo-myoglobin ions (j, product) were extracted (Supplemental Table S2) from the triplicate measurements, averaged and normalized. For each ∆**CV** setting one spectrum was generated (Figure 4) and analyzed semi-quantitatively by determining Gaussian fits for all molecular/supra-molecular ion species.

After recording mass spectra under increasing collision cell voltage difference settings (∆**CV**) and after determining Gaussian fits of charge structures for each ion series, the averaged **norm** (**educts**) values were plotted as a function of ∆**CV**,which resulted in a sigmoidal shaped course that represented the dependence of educt intensities (starting materials) on ∆**CV** settings (Supplemental Figure S1). All the y-values from the tangent line of the steep decline that fall within the 2dx interval around ∆**CV**<sup>50</sup> are used for the calculation of **ln k**# **mg** values which are then used in the Arrhenius plot (Supplemental Figure S2). As shown above at **Tcoll** = **Tamb** = **298 K** there applies ∆**CV** = 0 at which the value for **K**# **D m**0**g** is calculated (Table 1). **K**# **D m**0**g** is the apparent gas phase thermodynamic quasi equilibrium dissociation constants of heme loss of "neutral and resting" myoglobin in the gas phase. Then, ∆**G**# **m**0**g** is calculated using the van´t Hoff equation (Table 1).

–

∆ **Figure 4.** Nano-ESI mass spectra from myoglobin dissociation experiments. Different collision cell voltage differences (∆**CV**) are shown. (**A**) 4 V. (**B**) 30 V. (**C**) 60 V. (**D**) 120 V. Charge states and *m*/*z* values for selected ion signals are given for holo-myoglobin ions (red circles with dots on right ion series), apo-myoglobin ions (blue circles without dots on right ion series) and for the released heme ions (green dots on left ion series). Solvent: 200 mM ammonium acetate, pH 7.



(a) "neutral and resting" complex; (b) unitless number.

The gas phase dissociation reaction of RNAse S was investigated in the same manner. Upon electrospraying RNAse S which had been dissolved in 200 mM ammonium acetate solution, pH 7, the collision cell voltage difference was raised in a step-wise fashion and mass spectra were recorded (see the Methods section for in-solution handling steps). The ESI mass spectrum of RNAse S (Figure 5) provides ion series of multiply charged ions from which mean charge states of 6.4+ and average masses of 13,631.68 ± 0.20 Da and 13,544.23 ± 0.60 are calculated for the two most prominent RNAse S species. Commercial RNAse S represents two prominent protein complexes with clearly differentiated ion series, all of which represent related forms of RNAse S (Supplemental Table S3). For determining the overall apparent activation energy of the S-peptide dissociation reaction from RNAse S, all ion series were considered to equally represent the dissociation process as a whole, meaning that all ion signal intensities were subjected to normalization (Supplemental Table S4).

After recording mass spectra under increasing collision cell voltage difference settings (∆**CV**) and after determining Gaussian fits of charge structures for each ion series, the averaged **norm** (**educts**) values were plotted as a function of ∆**CV**, again generating a sigmoidal shaped course (Supplemental Figure S3). As before, all the y-values from the tangent line that fell within the **2dx** interval around ∆**CV**<sup>50</sup> were used for calculating **ln k** # **mg** values. These were subjected to draw the respective Arrhenius plot (Supplemental Figure S4). Then, the value for **K**# **D m**0**g** was calculated (Table 1) to represent the apparent gas phase quasi thermodynamic equilibrium dissociation constant

=

() ∆

of S-peptide loss from "neutral and resting" RNAse S in the gas phase. Finally, ∆**G**# **m**0**g** is calculated using the van´t Hoff equation (Table 1).

∆

 #

a) "neutral and resting" complex;

#

thermodynamic quasi equilibrium dissociation constants of heme loss of "neutral and resting"

**∆**

#

#

#

= =

#

− − − − #

∆

∆

**Figure 5.** Nano-ESI mass spectra from RNAse S dissociation experiments. Different collision cell voltage differences (∆**CV**) are shown. (**A**) 3 V. (**B**) 17 V. (**C**) 30 V. (**D**) 50 V. Charge states and *m*/*z* values for selected ion signals are given for RNAse S ions (red/purple diamonds on right ion series), S-protein ions (light blue/dark blue filled circles on right ion series) and for the released S-peptide ions (light green/dark green circles on left ion series). Solvent: 200 mM ammonium acetate, pH 7.
