Elucidation of isomeric structures for ubiquitin [M + 12H]12+ ions produced by electrospray ionization mass spectrometry.

Gas-phase deprotonation reactions, hydrogen-deuterium exchange reactions and collision-induced dissociation (CID) were used to distinguish between two isomeric forms of [M + 12H]12+ produced from the protein ubiquitin. Ions were generated by electrospray ionization and studied in a Fourier transform ion cyclotron resonance mass spectrometer. For [M + 12H]12+ formed directly from the electrospray process, deprotonation reactions with ammonia and 2-fluoropyridine yield non-linear pseudo-first-order kinetic behavior that indicates the presence of two ion structures. The fraction of ions that undergo the fastest deprotonation reactions, and is presumably the least energetically stable isomer, accounts for approximately 60% of the [M + 12H]12+ produced by electrospray. In reactions with D2O and CD3OD, the [M + 12H]12+ which are deprotonated faster exchange the first 11 +/- 1 hydrogens more readily that the remaining [M + 12H]12+ population. Results from CID experiments, obtained as a function of reaction time with the amines, also indicate the existence of more than one [M + 12H]12+ structure. The CID fragmentation patterns provide information about the general locations of the charge sites. Surprisingly, evidence for only one structure (the slow-reacting, more stable species) is found for [M + 12H]12+ that is produced by gas-phase deprotonation of [M + 13H]13+, which is the "fully protonated' form of ubiquitin. These results are discussed in terms of ubiquitin isomers related to protonation site and three-dimensional conformation.