Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 22, Issue 3, Pages 408-417Publisher
SPRINGER
DOI: 10.1007/s13361-010-0052-1
Keywords
Protein complex; Noncovalent interactions; Dissociation constant; Electrospray ionization; Biological mass spectrometry
Funding
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Canada Foundation for Innovation (CFI)
- Province of Ontario
- Canada Research Chairs Program
- University of Western Ontario
Ask authors/readers for more resources
Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K (d) values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine beta-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the direct ESI-MS approach is capable of providing K (d) values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 mu L min(-1) provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein-protein complexes in solution.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available