Supporting Information for: Real-time Investigation of the H/D Exchange Kinetics of Porphyrins and Oligopeptides by means of Neutral Cluster Induced Desorption/Ionization Mass Spectrometry A. Portz 1, C. R. Gebhardt 2, and M. Dürr 1, 1 Institut für Angewandte Physik, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany 2 Bruker Daltonik GmbH, D-28359 Bremen, Germany Corresponding author: michael.duerr@ap.physik.uni-giessen.de (M.D.) S1
This Supporting Information includes (I) Details on Monte Carlo simulations (II) Analysis and modelling of H/D exchange in angiotensin II (III) Analysis and modelling of H/D exchange in hexaglycine (IV) MS/MS experiments on H/D exchange in angiotensin II I Details on Monte Carlo simulations The H/D exchange reaction was simulated in discrete time steps t i. At every time step, each of the exchangeable hydrogen atoms at the respective molecule can be replaced with an exchange probability p f ; in the simulations this is realized by using a random number generator which generates a number between 0 and 1. If this number is below p f, the H atom is exchanged by the D atom. Accordingly, with a probability of 1 p f, the state remains undeuterated. Analogously, a D atom can be replaced by an H atom with a probability for this back reaction, p b, whereas the probability for the D atom being not replaced is 1 p b. In the simplest case, all exchangeable hydrogen and deuterium atoms in one molecule are exchanged with the same probability p f and p b, respectively. To simulate the kinetics of molecules with different functional groups, the whole entity of exchangeable H atoms can be subdivided into subgroups with the same exchange probabilities p f,i and p b,i in one subgroup i but different values of p f,i and p b,i for each subgroup. The relative intensity I rel,d for each degree of deuteration d can be derived for every time step by counting how often a d-fold deuterated molecule occurs during that time step divided by the total number of simulated molecules. The simulations were carried out with 2000 molecules and many different combinations of p i have been tested. The best result is considered to be the one which minimizes the value d i I rel,d,meas(t i ) I rel,d,sim (t i ) 2. With a fixed time step t i+1 t i = 3 s, the probabilities p f,i and p b,i can be converted into pseudo first-order rate constants k f,i and k b,i assuming a constant local concentration of H 2 O and D 2 O. S2
II Additional data on H/D exchange in angiotensin II In Fig. S1, the change of the relative signal intensities of selected degrees of deuteration with time is plotted (compare Fig. 8 in the main article) but in this case the data are compared to the best fit of Monte Carlo simulations taking into account only one single pair of rate constants. Apparently, no good fit could be achieved: the results of the simulations are too slow for the lower degrees of deuteration and too fast for the higher degrees of deuteration. This behavior is also reflected in the deviation between the simulated and Figure S1: Relative signal intensities of selected degrees of deuteration for angiotensin II (symbols + solid lines) as a function of time together with the corresponding results of Monte Carlo simulations (dashed lines). In contrast to the simulations shown in Fig. 8 in the main article, only one rate constant was used in the Monte Carlo simulations. Apparently, for the lower degrees of deuteration [d 11 in (a)], the change with time is slower than observed in the experiments but for higher degrees of deuteration [d > 14 in (b)], change with time is faster than observed in the experiments. S3
Figure S2: Logarithmic plot of 1 d/ d max indicating at least three different rate constants being operative. experimentally obtained dependence of the mean degree of deuteration on time [Fig. 7(e) in the main article]. The need for at least three different rate constants to properly describe the system is also obvious from Fig. S2, where the logarithm of the normalized mean degree of deuteration is plotted as a function of time. S4
III Data on H/D exchange in hexaglycine H/D exchange experiments in hexaglycine have been performed similar to the experiments with angiotensin II and the two porphyrins as described in the main article. In the case of hexaglycine, the singly charged negative ions [M-H] were analyzed. The experimentally determined change of the degrees of deuteration with time is shown in Fig. S3, highest degree of deuteration observed was dmax = 7 corresponding to the 5 H atoms at the amide groups of the molecule s backbone and the two H atoms at the terminal amine group (in total seven) as the proton of the terminal carboxylic acid group is detached in the case of the investigated [M-H] ions (Fig. S4). Direct comparison of, e.g., the intensity of d = 5 in the case of angiotensin II (Fig. 8) and Figure S3: Experimental results for the relative signal intensities of the 7 degrees of deuteration for hexaglycine (symbols + solid lines) as a function of time together with the corresponding results of the Monte Carlo simulations (dashed lines). (a) lower degrees of deuteration. (b) higher degrees of deuteration. S5
Figure S4: Structural formula of the negatively charged hexaglycine [M-H] with exchangeable hydrogen atoms printed in green. H atoms at the backbone are highlighted by a blue background, two further exchangeable H atoms are highlighted by an orange background. hexaglycine (Fig. S3) reveals the much slower exchange for the higher degrees of deuteration in hexaglycine when compared to the respective degrees of deuteration of angiotensin II. This is also reflected in the results of the according Monte Carlo simulations which yield a good fit with the experimental data when two rate constants are applied, a high one (similar to k 1 in angiotensin II) which applies to a subset of two functional groups, and a low one (similar to k 3 in angiotensin II), which applies to a subset of the remaining 5 functional groups. The values are listed in Tab. 1 in the main article. S6
IV MS/MS experiments on H/D exchange in angiotensin II MS/MS experiments were performed 24 h after the H/D experiments were stopped and the recipient was kept at the base pressure of 10 6 mbar for the whole time. Re-exchange of D by H atoms takes place but even after 24 h, still some deuterium atoms are present in the angiotensin II molecules (Fig. S5, inset). Isolation and collision induced dissociation (CID) was performed (Fig. S5, main panel) with the molecules contributing to the m/z = 524.8 peak, i.e., molecules carrying two isotopes, either two 13 13 C atoms, or two D atoms, or one C and one D atom. In Fig. S6, the distribution of peak intensities for the different isotopologues after CID is shown for the peak around m/z = 785.5, which is associated with the [Asp-Arg-Val-TyrIle-His+H]+ -fragment. In comparison with the distribution obtained from an undeuterated sample under otherwise identical conditions, as well as with the distribution expected from a statistical distribution of two 13 C atoms in the molecule but no additional D atoms present, a significantly higher contribution of the highest peak at m/z = 786.4 is observed, indicating one or two D atoms to be predominantly present in this fragment. Thus this fragment carries at least one functional group with slow H/D exchange rate. All fragments for which such Figure S5: MS/MS spectrum as obtained by collision induced dissociation after isolating the peak at m/z = 524.8. Inset: Isotope pattern before isolation and fragmentation. The sample was kept in the recipient for 24 h after the H/D exchange experiment, the mean degree of deuteration is d = 1.26. S7
Figure S6: Blow up of the m/z region around 785.5 with the data from Fig. S3 shown as red line. For comparison, the results of an MS/MS experiment with a non-deuterated sample (black line) as well as the peak intensities expected from a statistical distribution of 13 C atoms in the intact molecule (blue dots) are shown. a predominant presence of D atoms has been observed in this experiment are summarized in Tab. S1. These fragments cover the hole backbone of the molecule and the results are therefore compatible with our assignment of the lowest rate constant k3 to the amide groups of the peptide s backbone. q = D R V Y I H P F additional loss +1 +1 X X X X X X X +1 X X X X X X +2 X X X X X X +2 X X X X X X X COOH COOH OH NH2, OH Table S1: One- and twofold deuterated fragments of angiotensin II indicating functional groups of slow exchange rates within these fragments. A loss of additional functional groups is indicated in the last column. S8