In this instalment, we discuss radical ion chemistry as an increasingly important area of mass spectrometry (MS) development
and the application to bioanalysis. At the current stage, most of the research is performed by a small set of academic groups.
Given the unique capability offered by radical MS compared to the traditional studies on even-electron ions of analytes, it
is likely that these types of fundamental studies will attract more attention and even be commercialized in the near future.
Radicals are atoms, molecules, or ions that contain one or more unpaired valence electrons or an open electron shell. They
are, generally, highly reactive because of their need to convert themselves to more stable, even-electron species. Well-controlled
radical chemistry can provide the means for conducting some of the most difficult and delicate chemical transformations, like
converting RNA to DNA by ribonucleotide reductase, an enzyme with a radical as its catalysis centre. Radical chemistry can
be coupled with mass spectrometry (MS) to tackle traditionally challenging problems such as sequencing disulphide proteins
and determining C=C location in lipids. Here, we discuss this increasingly important area of MS development and its application
MS has been established as a powerful tool for qualitative and quantitative chemical analysis. Its principle of analysis involves
at least three crucial steps for extracting as much information as possible from an analyte: Forming ions from the analyte
molecule of interest, performing mass-to-charge ratio (m/z) analysis (so-called mass analysis), and perturbing ions to induce m/z changes. It typically relies on tandem mass spectrometry (MS–MS), a process in which ions are allowed to interact with neutral molecules, electrons, ions, electromagnetic waves, and
During the past several decades, these three steps have reflected significant advances. The development of various soft-ionization
methods permits the transformation of almost any molecule or chemical entity from its original physical state to gas-phase
ions with the least change to its structure. (Nevertheless, it is frequently necessary to add a charge carrier to the analyte,
facilitating MS analysis.)
Several types of mass analyzers, the component of mass spectrometers that separate ions according to their mass-to-charge
ratios, have been developed and commercialized. Instruments with time-of-flight (TOF) analyzers offer a fast speed of analysis.
Fourier transform ion-cyclotron resonance (FT-ICR) and orbital ion trap instruments offer high-performance mass measurement
and achieve a resolution in millions and sub-parts-per-million mass accuracy. Accelerator MS (AMS) instruments offer superb
dynamic range (108 –1015) for isotope analysis. Finally, some instruments could even be made portable, miniaturized for performing field analyses.
For tandem MS, many activation and dissociation options are available, including collision-induced dissociation (CID) and
electron-based excitation or dissociation methods. Clearly, MS has entered into a golden age of advanced development and can
be readily applied to solving complicated problems, whether in the areas of drug discovery, cancer research, environmental
monitoring, or exploring Mars.
Some of our most pressing questions about MS concern whether its usefulness has been fully exploited and, if not, what its
next major advance will be. Its history shows that innovation in instrumentation and inquiries in new gas-phase ion chemistry
often went side-by-side, providing the impetus for additional MS development. We believe the scope of gas-phase ion chemistry
is unlimited, that its exploration so far has been only marginal, and that it may therefore give rise to the next major MS
An area of increasing attention is the development of gas-phase, radical-ion chemistry for bioanalysis. Radical ions, which
consist of unpaired electrons, derived from biomolecules (peptides, proteins, lipids, and carbohydrates) are significantly
less investigated than even-electron ions. The available ionization methods do not lend themselves to directly forming the
radical ions of biomolecules, so additional reaction steps are required to effect the transformation from even-electron ions.
Several research groups have invested a major effort in developing methods to produce radical ions of biomolecules. Given
the coexistence of two reactive functional groups within one ion, a charge and a radical site, the chemistry of radical ions
is rich. It differs significantly from the chemistry of even-electron ions. This difference can be exploited to provide structural
information that complements the structural information gleaned from even-electron ions. A well-known example is the development
of electron-capture dissociation (ECD) and electron-transfer dissociation (ETD). These techniques provide rich structural
information for protein analysis that complements the information from CID. Thus, it substantially improves the capability
of protein identification and characterization.
Bio-radicals have been implicated as important intermediates in a wide variety of biochemical processes. At the molecular
level, they are associated with oxidative damage to proteins, DNAs, and lipids, which have been shown to be related to ageing,
neurodegenerative diseases, and cancers. Several classes of enzymes also use radicals for catalysis. For example, ribonucleotide
reductase uses a free radical (thiyl radical) for its catalytic activity. Interestingly, the radical storage centre is tens
of amino acid residues away from the catalytic centre. It is not clear, at this point, how the enzyme tightly regulates radical
reactivity along this long-range radical transfer process. In general, knowledge of the bio-radical species is constrained
by the limited techniques available to analyze these reactive intermediates. Studying the gas-phase chemistry of bio-radicals,
therefore, provides direct experimental evidence of their intrinsic chemical properties (reactivity, energetics, and structure),
which can help explain the fate, including intra molecular and intermolecular radical transfer, of bio-radical species after
they initially form.
The following discussions provide examples of methods of forming bio-radical ions of different classes of biomolecules and
how radical chemistry and MS can generate useful structural information.