Effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts.
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 sigma(54) activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg(2+), BeF(3)(-), and ADP, (at approximately 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.
Department of Chemistry, University of California-Berkeley, Berkeley, California 94720, USA.
Harry J
HJ
Sterling
Joseph D
JD
Batchelor
David E
DE
Wemmer
Evan R
ER
Williams
D000085:Acetates / Q000737:chemistry*; D001419:Bacteria / Q000737:chemistry; D002021:Buffers; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000085:Acetates; D002021:Buffers; D046912:Multiprotein Complexes; D051877:RNA Polymerase Sigma 54; C018824:ammonium acetate