Results of the mechanistic studies of free-electron based fragmentation methods addressing peptide and protein structure analysis with mass spectrometry (MS), electron capture dissociation (ECD) and electron ionization dissociation (EID), are presented. Series of model peptides, H-RAAAA-X-AAAAK-OH and H-RGGGG-X-GGGGK-OH, were rationally designed and studied to understand the role of a single amino acid residue on ECD process. The results show a direct correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity and radical stability of the central amino acid, X, in model peptides. Moreover, comparison of ECD PIA distributions for polyalanine- and polyglycine -based peptides, despite a substantial structure differences between them, demonstrates a high correlation (R=0.86) as well. Ion activation prior to ECD qualitatively preserves the PIA correlation with hydrophobicity, however redistributes the fragmentation channels. This redistribution can be rationalized in terms of: (i) further dissociation of product ions, due to the excess of ion internal energy; (ii) expansion of precursor ion conformational landscape reducing the conformational specificity in localization of unpaired electron and allowing competition of different reactive groups to induce the backbone cleavage. In particular, the competition between N-terminal R, C-terminal K and central X side chains to participate in formation of aminoketyl radicals was considered to explain the central residue specific formation of z5 and [z6-H] ions; and to explain the shift of branching ratio between c- and z-ions toward the formation of c-ions. Further studies were focused on the studying of free electron – ion interaction cross sections. The difference in low energy electron capture cross sections (CSs) of ECD product ions was found to correlate with ECD PIA pattern being a function of electron irradiation period in ECD. The developed experimental methodology and ECD kinetic model were applied to deduce the dissociation rate constants and electron capture CSs from ECD PIA values. The observed correlation between low energy electron capture CSs and ion mobility (IM) CSs provides interesting material for further studies. Finally, characterization of interaction between high energy (50-100 eV) electrons and protein ions in EID demonstrates that the EID cross sections are proportional (R = 0.98), and of the same order of magnitude, to the values of the IM CSs. The results support the feasibility of employing the EID MS for accurate estimation of the geometrical cross sections of gaseous biomolecular ions as a method of biomolecular structure analysis. To summarize, improved understanding in (i) radical chemistry beyond standard ECD parameters and (ii) electron – ion interactions with respect to the ion structure increases the quantity and quality of information possible to achieve by MS/MS, and, therefore, provides a strong basis for further improvements in peptide and protein structure characterization as well as understanding of energy absorption and relaxation process in biomolecules.