Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.
Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (</= 2 min) experiment, providing 3D- architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)-mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI- TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein beta(2)-microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
David P
DP
Smith
Tom W
TW
Knapman
Iain
I
Campuzano
Richard W
RW
Malham
Joshua T
JT
Berryman
Sheen E
SE
Radford
Alison E
AE
Ashcroft
D002138:Calibration; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D010455:Peptides; D011506:Proteins