Chemistry Reference
In-Depth Information
(m/z)
2
=
(MW
+
z
2
H)/z
2
(7.2)
As
z
1
and
z
2
are adjacent charge states then
z
2
=
z
1
+
1. This relationship allows Equations
(7.1) and (7.2) to be combined to give
z
1
=
[
(m/z)
2
−
H
]
/
[
(m/z)
1
−
(m/z)
2
]
(7.3)
and
MW
=
z
1
(m/z)
1
−
z
1
H
(7.4)
Although it is trivial to solve these equations for simple peptides or a pure small
protein, deconvoluting a mass spectrum to determine molecular weights and relative
abundances of various components rapidly becomes intractable for manual calculations
when working with larger proteins or mixtures of proteins. All modern ESI-equipped
mass spectrometers include sophisticated deconvolution software derived from the above
mathematical relationships to search automatically and routinely for peaks that are from
the same molecular species but differ by charge state. The intensities of all related
peaks are added together and converted to a single
1 or zero charge state to pro-
duce the 'deconvoluted' spectrum revealing the protein molecular weight (Figure 7.6c).
Calculations generally require only a fraction of a second for even fairly complicated
mass spectra.
+
7.8.2 ESI-MS and Noncovalent Ligand-Protein Interactions
The screening of targets against ligands by mass spectrometry requires that the experi-
mental parameters of the ESI-MS process be adjusted such that the noncovalent interactions
between the ligand and target are maintained during both the desolvation process and
in the gas phase. ESI-MS has been used extensively to study proteins and complexes
of proteins with naturally occurring substrates, inhibitors and drugs. An extensive lit-
erature on this topic (more than 300 publications and numerous review articles, as
pointed out in a recent review
[
49
]
) indicates that although there are pitfalls, it is gen-
erally straightforward to adjust the parameters of the ESI-MS measurement so that the
signals measured in the mass spectrometer for a protein, its ligands and the noncova-
lent complexes thereof reflect the equilibrium concentrations of these species in solution.
The possibility that transient, nonspecific protein-ligand interactions may be maintained
in the desolvation process and observed in the mass spectrometer (thus generating false
positives) has been raised as an objection against the use of mass spectrometry in screen-
ing applications. However, there is now abundant evidence that the complexes that are
observed in the mass spectrometer do indeed reflect the binding constants in solution
as the conditions required for complete desolvation of the protein and specific protein-
ligand complexes ensure that the weaker complexes resulting from nonspecific interactions
are not maintained in the gas phase and are not observed in the mass spectrum.
[
49
]
To
confirm correct ESI-MS parameter adjustment, parameters for the ESI-MS measure-
ment are generally first optimized on a target with a ligand of known binding constant
such that the known binding constant is observed in the results. Early work on screen-
ing of inhibitors against carbonic anhydrase showed that multiple inhibitors could be
