The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.
Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.
Bruce
J E
JE
Environmental and Molecular Sciences Laboratory, The Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Smith
V F
VF
Liu
C
C
Randall
L L
LL
Smith
R D
RD
D001426:Bacterial Proteins / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D018832:Molecular Chaperones / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D008024:Ligands; D018832:Molecular Chaperones; C057842:SecB protein, Bacteria