Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry
Benson, L. M., Kumar, R., Cavanagh, J., & Naylor, S. (2003). Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry. Rapid Communications in Mass Spectrometry, 17(4), 267-271. DOI: 10.1002/rcm.903
The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (muESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/muESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D-28K. Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F. Copyright (C) 2003 John Wiley Sons, Ltd.