Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry.
The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.
Smith
David P
DP
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
Giles
Kevin
K
Bateman
Robert H
RH
Radford
Sheena E
SE
Ashcroft
Alison E
AE
D045304:Cytochromes c / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000737:chemistry*
D001613:beta 2-Microglobulin; D045304:Cytochromes c