wave heights in the region of 5–25 V and wave velocities between 200–400 m‧s
À1 are typically
used to achieve optimal separation. Further details of these parameters are beyond the scope
of this protocol. The reader is referred to other published methods for more information
(Ruotolo et al. 2008).
3. Collisional cross section calibration and comparison with structural data
• ESI-IM-MS spectra of calibrant proteins must be acquired under the same instrument conditions used for the MP/APol complexes for all parameters downstream of
the trap ion guide. When using native protein standards, the trap collision energy and
cone voltages should be decreased, relative to those needed to observe MP/APol
complexes, to prevent protein unfolding.
The calibration of CCSs from travelling wave IM ATD data is well established, and the CCSs
of the calibrant proteins are known. The reader is referred to several recent publications for
more information on the calibration procedure, which is now relatively routine (Ruotolo et al.
2008; Bush et al. 2010).
The power of IM lies in the ability to compare experimentally determined CCS data with those
of models or high-resolution structures. Several software packages, which use different
methodologies, have been developed to determine in silico CCS values for comparison with
IM data. The most reliable CCS calculation method is the trajectory method, which is
implemented in the software MOBCAL and other packages (see Mesleh et al. 1996; Shvartsburg
and Jarrold 1996, and MOBCAL–A Program to Calculate Mobilities. http://www.indiana.edu/
~nano/software/). Recent work has demonstrated the use of alternative, less computationally
intensive approaches that can provide data of similar quality, e.g. the corrected projection
approximation implemented in IMPACT (Marklund et al. 2015) or the projection superposition
approximation (Bleiholder et al. 2011).
14.5.4 Outlook
MS-based methods, including native MS, but also fast photochemical oxidation (FPOP)-MS, hydrogen deuterium exchange (HDX)-MS, chemical crosslinking-MS, and other footprinting approaches,
are becoming increasingly used to study membrane protein structure. We and others have
demonstrated the utility of nonconventional surfactants to solubilize MPs of both β-barrel and αhelical structures for native ESI-IM-MS (Hebling et al. 2010; Leney et al. 2012; Hopper et al. 2013;
Calabrese et al. 2015; Lu et al. 2016; Marty et al. 2016; Watkinson et al. 2017). We have also
performed a systematic analysis into the utility of a range of amphipols (A8-35, A8-75, A34-35,
A34-75, SAPol, and NAPol) for ESI-IM-MS analysis (Watkinson et al. 2015). Continued development, application, and refinement of novel solubilization methods, including the use of APols, for
native MS and other structural MS approaches will open new avenues to gain insights into the
functional mechanisms of a range of MPs.
Protocol prepared by Antonio N. Calabrese, Thomas G. Watkinson, Sheena E. Radford, and
Alison E. Ashcroft, Astbury Centre for Structural Molecular Biology, School of Molecular and
Cellular Biology University of Leeds, based on Calabrese et al. (2015), and Watkinson et al. (2015),
with comments added.
654
14 The Use of Amphipols in Mass Spectrometry
À1 are typically
used to achieve optimal separation. Further details of these parameters are beyond the scope
of this protocol. The reader is referred to other published methods for more information
(Ruotolo et al. 2008).
3. Collisional cross section calibration and comparison with structural data
• ESI-IM-MS spectra of calibrant proteins must be acquired under the same instrument conditions used for the MP/APol complexes for all parameters downstream of
the trap ion guide. When using native protein standards, the trap collision energy and
cone voltages should be decreased, relative to those needed to observe MP/APol
complexes, to prevent protein unfolding.
The calibration of CCSs from travelling wave IM ATD data is well established, and the CCSs
of the calibrant proteins are known. The reader is referred to several recent publications for
more information on the calibration procedure, which is now relatively routine (Ruotolo et al.
2008; Bush et al. 2010).
The power of IM lies in the ability to compare experimentally determined CCS data with those
of models or high-resolution structures. Several software packages, which use different
methodologies, have been developed to determine in silico CCS values for comparison with
IM data. The most reliable CCS calculation method is the trajectory method, which is
implemented in the software MOBCAL and other packages (see Mesleh et al. 1996; Shvartsburg
and Jarrold 1996, and MOBCAL–A Program to Calculate Mobilities. http://www.indiana.edu/
~nano/software/). Recent work has demonstrated the use of alternative, less computationally
intensive approaches that can provide data of similar quality, e.g. the corrected projection
approximation implemented in IMPACT (Marklund et al. 2015) or the projection superposition
approximation (Bleiholder et al. 2011).
14.5.4 Outlook
MS-based methods, including native MS, but also fast photochemical oxidation (FPOP)-MS, hydrogen deuterium exchange (HDX)-MS, chemical crosslinking-MS, and other footprinting approaches,
are becoming increasingly used to study membrane protein structure. We and others have
demonstrated the utility of nonconventional surfactants to solubilize MPs of both β-barrel and αhelical structures for native ESI-IM-MS (Hebling et al. 2010; Leney et al. 2012; Hopper et al. 2013;
Calabrese et al. 2015; Lu et al. 2016; Marty et al. 2016; Watkinson et al. 2017). We have also
performed a systematic analysis into the utility of a range of amphipols (A8-35, A8-75, A34-35,
A34-75, SAPol, and NAPol) for ESI-IM-MS analysis (Watkinson et al. 2015). Continued development, application, and refinement of novel solubilization methods, including the use of APols, for
native MS and other structural MS approaches will open new avenues to gain insights into the
functional mechanisms of a range of MPs.
Protocol prepared by Antonio N. Calabrese, Thomas G. Watkinson, Sheena E. Radford, and
Alison E. Ashcroft, Astbury Centre for Structural Molecular Biology, School of Molecular and
Cellular Biology University of Leeds, based on Calabrese et al. (2015), and Watkinson et al. (2015),
with comments added.
654
14 The Use of Amphipols in Mass Spectrometry