Solution-phase chelators for suppressing nonspecific protein-metal interactions in electrospray mass spectrometry.
Protein-metal complexes may be transferred from solution into the gas phase by electrospray ionization (ESI), such that they can be directly analyzed by mass spectrometry (MS). In principle, therefore, ESI-MS represents a simple and elegant approach for gaining insights into the binding stoichiometry and affinity of these assemblies. Unfortunately, the formation of nonspecific metal adducts during ESI can be a severe problem, often leading to binding levels that are dramatically higher than those in bulk solution. Focusing on several calcium binding proteins as test systems, this work explores the suitability of different salts to serve as metal source. Despite their widespread use in previous ESI-MS studies, calcium chloride and acetate induce extensive nonspecific adduction. In contrast, much lower levels of artifactual metal binding are observed in the presence of calcium tartrate. In the case of high and intermediate affinity proteins, the resulting ESI-MS data are in excellent agreement with the calcium binding behavior in bulk solution. The situation is more challenging when studying proteins with very low affinities, but in the presence of tartrate qualitative information on protein-metal interactions can still be obtained. The beneficial effects of tartrate also extend to zinc binding experiments. This work does not directly explore the mechanism by which tartrate suppresses nonspecific metalation. However, it seems likely that weak chelators such as tartrate sequester metal ions within rapidly shrinking droplets during the final stages of ESI, thereby reducing nonspecific metal adduction to protein carboxylates. The use of tartrate and possibly other weak chelators will greatly enhance the reliability of future ESI-MS studies on the interactions of proteins with divalent metal ions.
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Jingxi
J
Pan
Kun
K
Xu
Xiaoda
X
Yang
Wing-Yiu
WY
Choy
Lars
L
Konermann
D016477:Artifacts; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013644:Tartrates / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D008670:Metals; D013644:Tartrates; D015032:Zinc; D002118:Calcium; C029768:tartaric acid