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% ND3 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 ND3 was injected into a radio-frequency quadrupole. In the absence of ND3 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.

Two-dimensional decomposition of H-D exchange mass spectra of multicharged ions of biopolymers and their separation into components with independent H-D substitutions.

The work is aimed at developing a numerical method for analysing mass spectra of deutero-substituted multicharged ions of biopolymers to determine contributions of components presumably corresponding to different biomolecule conformations. The two-dimensional decomposition of the H-D exchange mass spectra of two, three and four charged apamin ions with their separation suggests that the reaction of apamin ions with ND3 molecules in the gas phase reveals hypothetically three different structural modifications of apamin ions. Usually for H-D exchange mass spectra, the presence of many resolvable protein structures was determined from measured distributions of peak intensities of ions with the same charge state. The method is new and has no published analogues. Graphical abstract.

Two possible improvements for mass spectrometry analysis of intact biomolecules.

The goals of our study were to investigate abilities of two approaches to eliminate possible errors in electrospray mass spectrometry measurements of biomolecules. Passing of a relatively dense supersonic gas jet through ionization region followed by its hitting the spray of the analyzed solution in low vacuum may be effective to keep an initial biomolecule structure in solution. Provided that retention of charge carriers for some sites in the biomolecule cannot be changed noticeably in electrospray ion source, decomposition and separation of charge-state distributions of electrosprayed ions may give additional information about native structure of biomolecules in solution.

Decomposition of charge-state distributions for a better understanding of electrospray mass spectra of bioorganic compounds. Part 1: basic formalism.

Further development in our approach for the interpretation of a mass spectral peak series resulting from unique modifications at a number of molecular sites is described and its application for the analysis of electrospray mass spectra of multiply-charged ions of biopolymers is specified. This method was based initially on the assumption that the modifications are mutually independent. The output is a set of modification probabilities for the considered molecular sites, which explains the observed peak intensity distribution with the best accuracy. It is shown here that the method is also applicable, in some cases, to the strong interaction between modified sites. This is important for the formation of multiply-charged ions, at least in the gas phase, since it should be significantly influenced by the internal electric fields. The capabilities and limitations of this approach are discussed. One limitation is that only unimodal distributions (those having only one maximum) are possible in the considered model. Another is that relatively large biomolecules may not be suitable for this type of analysis. Some experimental results of the application of this method are described in Part 2 of this work.

Decomposition of charge-state distributions for better understanding of electrospray mass spectra of bioorganic compounds. Part 2: application of the method.

The results of the analysis of electrospray mass spectra of b-endorphin and chicken egg lysozyme in different conditions of data acquisition using the method described in Part 1 of the work are reported. At least partial unfolding during the process of ion formation in the electrospray ion source of an initially native biomolecule of lyzozyme in solution should supposedly explain the received set of probabilities of proton retention by basic and acidic residues of this molecule for all considered conditions of data acquisition.