Biology Reference
In-Depth Information
Using these kinetic experiments, two different processes can be monitored to provide
an estimate of the kinetics of the covalent reaction once the compound is noncova-
lently bound to the binding site. The first experiment measures the number of binding
sites that have not yet reacted and therefore are available for Flutax-2 binding. This
measures the kinetics of the covalent reaction. In the second kinetics measurement,
MTs are pelleted and the concentration of unreacted compound measured, such that,
both covalently and noncovalently reacted compounds are pelleted. This measure-
ment accounts for the kinetics of the noncovalent binding reaction. If the covalent
reaction between the compound and the sites is immediate, such as with ZMP, no
difference will be observed between the two measurements. In this case, the covalent
reaction immediately follows noncovalent binding. However, if the covalent reaction
is slow, as with dactylolide, and the noncovalently bound compound can exchange
with unbound compound, a significant difference between measurements will be
obtained. This indicates a significant delay between the noncovalent and the covalent
reaction of dactylolide (
Fig. 19.4
;
Field et al., 2012
).
19.2.6
Mass spectrometry
To determine where on the protein covalent binding is occurring, a detection method
for the ligand bound to the protein is used. Mass spectrometry (MS) is the most suit-
able method to characterize ligand-protein covalent binding. Specialized instru-
ments based on hybrid configurations allow the detailed analysis of covalently
modified peptides. MS is also used to determine the modified amino acid residue
(s) at which the ligand is binding by the comprehensive analysis of the corresponding
fragmentation spectra, giving direct evidence of the binding site (
Buey et al., 2007;
Calvo et al., 2012; Field et al., 2012
).
To determine the modified peptides, first characterize the fragmentation spec-
trum of the MSA by MS. This information is crucial to determine the putative diag-
nostic ion(s) for subsequent ion-filtering experiments, mainly performed in triple
quadrupole systems. Precursor ion scanning (PIS) and selective reaction monitoring
(SRM) allow the selection of a given subset of peptides according to their masses
and the presence of specific ions in the fragmentation spectrum when collision
dissociation is induced. Thus, only those ions that satisfy both conditions are ana-
lyzed, whereas the rest of the ions are deflected. In the hybrid systems, it is possible
to combine the benefits from a conventional triple quadrupole system and a linear
ion trap. Ions filtered by PIS or SRM are detected with improved resolution to de-
termine mass-charge of the precursor ion and the corresponding fragmentation spec-
trum. The comprehensive analysis of the fragmentation spectra can determine not
only the masses of the MSA-attached peptides but also the modified residue(s) within
the sequence.
In a normal experiment to characterize the peptides/residues modified by an
MSA, the ligand is first bound to the protein and the samples are subjected to diges-
tion using a protease. The tryptic peptide mixtures for ion-filtering experiments are
then subjected to liquid chromatography coupled to hybrid MS (LC-MS). Peptides
