Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

162 Doherty-Kirby and Lajoie
metal ion partitioning and the sequence of metal binding. To date, the determination
of stoichiometry of calcium binding is the most documented application
of ESI-MS in the study of metal-binding proteins.
Standard “denaturing” ESI-MS conditions used for determination of protein
molecular weight are solutions of pH 2.0 to 4.0 in the positive ion mode and 8.0
to 10.0 in the negative ion mode to yield the best sensitivity. An organic cosolvent,
such as acetonitrile or methanol, is often used to enhance sensitivity and signal
stability. To study protein interactions by ESI-MS, nondenaturing conditions
including a volatile buffer, such as ammonium acetate or ammonium carbonate
close to neutral pH, and low temperature are used. These milder conditions lead
to a significant decrease in sensitivity. Mass spectrometer parameters, such as
capillary temperature, ion mode, and voltage, must be optimized for each system.
This requires a balance between maintaining the intact complex while
adjusting parameters for sufficient ion desolvation and ionization.
A set of criteria should be met to provide evidence that the interactions
observed are specific (4). First, for tight binding complexes, the predominant
species should correspond to that identified for the protein in solution. The intensity
of signal corresponding to complexes should be altered by changes in
instrumental conditions, such as increased capillary temperature or applied voltage.
Mass spectra should reflect differences in complex formation and be sensitive
to solution conditions, such as pH, temperature, type and concentration of
buffer components. Structural modification of the complex components either
in the protein or the ligand should alter the relative intensity of observed species
corresponding to increased or decreased binding in solution. (4).
This chapter is a short review of studies on noncovalent interactions of calcium-binding
proteins that have been characterized by ESI-MS, followed by
some detail of our own work on calmodulin (CaM). The studies of calcium-binding
proteins in their native state using ESI-MS can be grouped into three types:
1. Properties of calcium binding (i.e., stoichiometry and cooperativity) determined
through examination of the metal-bound species present in mass spectra and comparison
to spectra of the metal free protein.
2. Conformational changes detected by a shift in the mass-to-charge (m/z) envelope
or with the use of hydrogen–deuterium exchange.
3. Interactions of calcium-binding proteins with other molecules.
CaM is, by far, the most extensively studied Ca 2+ -binding protein. A number
of groups have determined by ESI-MS that CaM binds four calcium ions, consistent
with the observed stoichiometry in solution (3,5–8). Studies with calcium,
magnesium, and terbium also showed that there were additional
low-affinity binding sites (7).