The novel mass spectrometric technique tandem ionization mass spectrometry (TIMS) employs irradiation of gas-phase even-electron molecular ions of both polarities with >10 eV electrons. This leads to the production of radical molecular cations and anions of large biomolecules. The parent even-electron ions are produced by laser desorption, matrix-assisted laser desorption/ionization (UV and IR) or electrospray ionization and trapped in the cell of a Fourier transform mass spectrometer before irradiation with electrons. For multiply-charged polypeptide cations (up to 16+ for cytochrome c) and di-anions, TIMS produced radical [M + nH]( n + 1)+• cations and previously unreported [M − 2H]−• anions, respectively. Subsequent collisional activation of these species, in contrast to their even electron counterparts, gave only small neutral losses (mainly CO2) regardless of the ionic charge, polarity or lability. This process was rationalized through intramolecular hydrogen atom transfer in cations. Measurements of the threshold energies for electron ejection has now been extended to the protonated porphyrin C76H94N4 [ IE(MH+) = 12.8 ± 0.3 eV] and to multiply-charged polypeptide cations and anions. Serendipitously, it was found that, in the absence of electrons, [M + nH]( n + 1)+• polypeptide cations can also be formed in energetic collisions during the ion isolation process in Fourier transform mass spectrometry.