REACTIVE SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 12

Most of the labeling of peptides and proteins has been performed using ROS,
particularly hydroxyl radicals [87]. The detailed chemistry of reactive halogen,
nitrogen, and oxygen species-mediated oxidation of side chains is described in
Chapters 3-5.
1.2.1.4 Hydroxyl Radical Labeling. The probing of the solvent-accessible
surface area (SASA) of proteins using reactions with • OH radical and
subsequent identification of oxidized sites by MS has been widely used for the
oxidative labeling of proteins [86, 87, 120-129]. Because of the low specificity
of the • OH radical, a number of target sites in the protein are modified, result-
ing in an incorporation of oxygen atoms into amino acid side chains. This yields
characteristic +16 Da adducts in the mass spectrum. The oxidative labeling of
the target sites in proteins is influenced by intrinsic reactivity and solvent
accessibility [87, 130]. Hydroxyl radicals preferentially react with sulfur-
containing heterocyclic and aromatic amino acids with rate constants varying
from 5 × 10 −9 to 1 × 10 10 /M/s [1, 12, 131-134]. However, α-carbon hydrogen
atoms, such as those found in Gly and Ala, have been found to react slower
with the • OH radical ( k ∼ 10 9 /M/s) [131]. More is discussed in Chapter 4. The
reactivity of amino acid residues with • OH radicals and the ability of MS to
detect oxidized products under aerobic conditions have the potential to probe
the structure of proteins. The primary oxidation products for the amino acid
side chains are presented in Table 1.3 [87, 135, 136]. Various methods of • OH
generation have been described, including Fenton chemistry, electrochemistry,
coronal discharge methods, photochemistry, radiolysis, and pulse electron
beams (Chapter 4) [87, 87, 137, 138].
A scheme of • OH radical probing of the protein is presented in Figure 1.5
[87]. In this approach, the side chains of the protein and its complexes are
modified using • OH radicals, followed by X-ray exposure and digestion with
proteases [82]. Quantification by MS determines the extent of modification. A
tandem MS technique is applied to assess particular modified sites. This quan-
titation provides information about the solvent accessibility of each peptide
in both the isolated and complexed states of the protein. The slower rate of
binding proteins compared to the free protein indicates the influence of the
binding process on the peptide containing the reactive side chains. Further-
more, allosteric changes in the conformation during binding may also produce
an increase in reactivity [87]. This phenomenon can be observed in dose-
response experiments. The use of a freeze-drying technique for the removal
of H 2 O 2 to determine the conformation of a protein was demonstrated to be
unsuitable due to the oxidation of proteins under cold conditions [139].
There has been recent focus on the duration of either electron or laser
pulses to the protein in order to prevent structural equilibrium during the
timescale of exposure [140-143]. The use of timescales in submicroseconds has
been determined for oxidative protein surface mapping [140, 142, 143]. A
continuous-flow capillary setup has also been employed with an objective of
exposing individual molecules of a protein to only a single • OH pulse [123,