2013 Volume 2 Issue Special_Issue Pages S0004
Peptide radicals play a significant role in biology as well as mass spectrometry. They can be differentiated into two groups: conventional hydrogen-deficient radicals, e.g. M+• as in electron ionization, and much more rare hydrogen-abundant radicals, e.g. [M+2H]+•, as in electron capture/transfer dissociation. The dissociation chemistries of these two types of radicals are vastly different. Both types tend to lose small molecules or radical groups, but the overlap between the losses from different radical types is minimal. The backbone cleavage for hydrogen-deficient radicals is dominated by Cα–C cleavage (a•, x fragments) and for hydrogen-abundant radicals—by N–Cα cleavage (c, z• ions). The latter types of fragmentation produces more sequencing information than the former. Therefore, hydrogen-abundant peptide radicals are more valuable in mass spectrometry. The efficiency of the main method of their production, electron capture/transfer dissociation, is however limited by charge reduction. Alternative methods of generation of hydrogen-abundant radicals are needed to improve the sequencing capabilities of mass spectrometry.
IUPAC Gold book (http://goldbook.iupac.org) defines radical ion as “a radical that carries an electric charge.” Following this definition, we will not differentiate between neutral radicals and radical ions, calling both species simply radicals. Indeed, neutral radicals can be zwitterions, and ionic radicals may acquire their charge by attaching a charged adduct, e.g. a proton, Na+ or Cl−. Acquiring charge often does not interfere with the radical nature of the species. Similarly, peptides and proteins in biology are called simply peptides and proteins, although almost all of them in native conditions exist in ionized state.
Peptide radicals play a significant role in biology as well as in mass spectrometry.1,2) But all peptide radicals are not created equal; instead, they can be separated into two groups. The first group is known in mass spectrometry at least since 1958.3) At that time, electron ionization (EI) was the dominant method of obtaining molecular ions:
 | (1) |
The radicals M+• are called hydrogen-deficient,1,4) because they are lacking a hydrogen atom to become a protonated molecule, which is “in balance” with its 1+ charge state:
 | (2) |
To the same class of hydrogen-deficient species belong the radicals produced from protonated peptides by UV photoexcitation,5) collisions with oxygen,6) fragmentation of a modified peptide,7) or peptide-ion complex,8,9) or EI10):
 | (3) |
or electron detachment from deprotonated molecules9,10):
 | (4) |
Hydrogen-deficient radicals are not necessarily charged. According to the IUPAC Gold book’s definition, alkyl radical “are carbon-centered radicals derived formally by removal of one hydrogen atom from an alkane, e.g. CH3CH2C⋅H2 propyl.” Thus, alkyl radicals are hydrogen-deficient radicals in a very direct sense.
On the other hand, there are hydrogen-abundant radicals, i.e. species that cannot be obtained from molecules by hydrogen abstraction. These are, e.g. the charge-reduced, intermediate species in electron capture/transfer dissociation (ECD/ETD11,12)):
 | (5) |
While the difference between the hydrogen-deficient and hydrogen-abundant radicals in the same charge state consists only in one hydrogen molecule, H2, the dissociation chemistries of these two types of radicals are vastly different. Both tend to lose small molecules or radicals, but the overlap between the losses from different radical types is minimal.13,14)
The backbone fragmentation of hydrogen-deficient radicals is dominated by Cα–C cleavage (a•, x fragments), especially for not so large molecules.10,13,15,16) At the same time, backbone fragmentation of hydrogen-abundant radicals is dominated by N–Cα cleavage (c, z• ions).11,12) An interesting, and yet unexplained feature of hydrogen-abundant radical cations is that they do not usually lose H2 to become hydrogen-deficient radicals. Although the latter species are more stable, they do exhibit H2 loss—for instance, such a loss is observed from z• ions with N-terminal Ser or Thr residue.17) Among the mechanisms of conversion of hydrogen-abundant to hydrogen-deficient species, the most important is fragmentation,14) but rearrangement is also possible.18)
Which type of radicals provides more sequencing information? A recent study by Coon’s group gives the answer to this question: in “negative ETD” (technique similar to electron detachment dissociation, EDD,19) that produces hydrogen-deficient anion intermediates) of a peptide mixture, they identified three times fewer peptide sequences compared to ETD in the positive ions mode of the same peptide mixture.20) Even after taking into account the possible difference in ionization efficiency in positive and negative ion mode, one comes to the conclusion, supported by a multitude of complementary observations, that fragmentation of hydrogen-abundant radicals is far more informative than dissociation of hydrogen-deficient radicals. The problem is that, besides ECD and ETD, there are few sources of these radicals.
To some extent, the problem may be alleviated when the two worlds are united and hydrogen-deficient radicals capture an electron, as in electron ionization dissociation (EID13)). A series of events occur when protonated molecules acquire a radical charge (a charge site associated with an unpaired electron): first, within a few femtoseconds, the newly acquired charge moves along the backbone to find its position on a group with low ionization energy21) and away from the ionizing protons.5) If the residing place of the charge is the N-terminus, a labile hydrogen atom originating from a carboxylic group (e.g. from the C-terminus) or from the phenolic moiety of the tyrosine residue, moves along the backbone to create a protonated N-terminus.21) Now in the ion, all charges are defined by ionizing protons, and the radical is residing elsewhere. When such a distonic ion captures an electron, it becomes a diradical, with one of the two possible outcomes. Either the radicals quench each other, and the formed species fragments according to even-electron rules, or an ECD-type fragmentation occurs. Recent results by Kalli and Hess22) indicate that both processes occur in a competitive manner. For not-so-large peptides, the first process appears to dominate, but EID results for larger polypeptides indicate that, for them, the second (more desirable) outcome becomes prevalent. Still, EID, where precursor ions are first ionized, often multiple times,23) and then capture an electron and fragment, is plagued by low overall efficiency when performed in a Penning trap of the ion cyclotron resonance (ICR) mass spectrometer. To achieve a boost in fragmentation efficiency, EID should be performed in a device with a larger trapping ability (the axial potentials in an ICR trap are often only 0.5 V or lower, especially during detection). Designing such a device may not be so easy, as fast electrons disturb the precursor ion cloud, and ion cooling by collisions with neutral gas may be required, while avoiding ionization of that gas. Alternatively, novel methods of generation of hydrogen-abundant radicals may be required for improving the sequencing ability of mass spectrometry.
Peptide radical cations come in two kinds, with hydrogen-abundant radicals being more useful for sequencing than their hydrogen-deficient counterparts. So far, electron capture remains the prime production mechanism of the former species, although, in principle, other pathways should also be possible. Thus, further instrument and method development is required to boost the performance of mass spectrometry in polypeptide sequencing.
This work was supported by the Knut and Alice Wallenberg Foundation, European Union (consortium EPITOPE), VINNOVA Foundation, CancerFonden as well as the Swedish Research Council.
