Separation of mass spectra of hydrogen-deuterium exchanged ions obtained by electrospray of solutions of biopolymers with unknown primary structure.

The work is dedicated to further development of our described method for analyzing mass spectra of biomolecules acquired as a result of hydrogen-deuterium exchange reactions (HDXs). The modified method consists of separating HDX distributions via their approximations by a minimum number of components corresponding to independent H/D substitutions and independent charge carrier retentions in different spatial isoforms or conformations of biomolecules with unknown primary structures. In this case, neither the natural isotopic distribution nor the exact number of active sites involved in HDXs and H or D attachments can be determined in advance. Original H/D electrospray mass spectra of an apamin solution were taken from our previous work. In that work, taking into account the natural isotopic distribution of apamin molecules, three main conformations of apamin ions were found as a result of separating the H/D mass spectra of the apamin solution for the gas flow with the addition of about 10% ND molecules. Using the proposed modified method that does not require knowledge of the primary structure of the biomolecules gave similar results with slight deviations of calculated HDX distributions of the apamin ions from those obtained earlier. The maximum difference between mean values of the calculated HDX distributions for ions of the same charge in both cases does not exceed a few percent. In addition, HDX mass spectra of the apamin complex with an adduct of unknown structure were processed. Such analysis gave also three main fractions of ions with relatively large contributions when ND was injected into a radio-frequency quadrupole. In the absence of ND flow, the results of calculations for apamin and its complex were close to each other too. The formation of the apamin complex most probably in solution was confirmed by performed calculations.