Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 21, Issue 6, Pages 1045-1049Publisher
SPRINGER
DOI: 10.1016/j.jasms.2010.02.003
Keywords
-
Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM062163, R01GM064712] Funding Source: NIH RePORTER
- NIGMS NIH HHS [R01 GM064712, R01 GM064712-08, R01 GM062163, R01-GM062163, R01-GM064712-08] Funding Source: Medline
Ask authors/readers for more resources
Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a 054 activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg2+, BeF3-, and ADP, (at similar to 60 degrees C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration. (J Am Soc Mass Spectrom 2010, 21, 1045-1049) (C) 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry
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