14.5 Protocol 14.1: Characterization of Amphipol-Trapped Membrane
Proteins by Electrospray Ionization-Ion Mobility Spectrometry-Mass
Spectrometry
14.5.1 Introduction
Non-covalent (or “native”) mass spectrometry (MS) is emerging as an invaluable method to study
membrane proteins (MPs) and MP complexes, as well as their interactions with lipids and small
molecules (Laganowsky et al. 2014; Bechara et al. 2015; Bechara and Robinson 2015; Konijnenberg
et al. 2015; Rajabi et al. 2015; Gault et al. 2016). The approach requires samples to be maintained in
their native conformations in solution before ionization, typically by the use of electrospray ionization
(ESI). Implementation of non-covalent ESI-MS with ion mobility (IM) spectrometry, i.e. ESI-IM-MS,
allows measurement of the ions’ rotationally averaged collisional cross sections (CCSs), together with
their mass-to-charge (m/z) ratios, in a single experiment. Ion CCSs are related to shape, size, and
charge, and can yield structural information about biomolecules (Ruotolo et al. 2008).
Typically, MP analysis using ESI-IM-MS has been performed by solubilizing the protein of
interest with detergents and subsequently dissociating the ionized MP/detergent complex in vacuo by
collisional activation, leading to the ejection of the MP from its solubilizing media for mass analysis
(Barrera et al. 2008; Laganowsky et al. 2013). This is key to successful MS analyses, as MP/surfactant
complexes are highly heterogeneous, and therefore measurement of a complex’s intact mass is not
possible. We, and others, have developed detergent-free methods for the study of MPs using ESI-MS,
by introducing MPs solubilized with amphipols (APols), bicelles, or nanodiscs into the gas phase for
interrogation (Hopper et al. 2013; Calabrese et al. 2015; Watkinson et al. 2015; Marty et al. 2016a).
Here, a general approach is described for the study of APol-solubilized MPs using ESI-IM-MS,
highlighting key stages of the sample preparation and MS analyses that must be optimized for each
system.
In the following, the protocol itself is printed in roman, background and comments in italics,
preceded by a pointing hand ().
14.5.2 Materials
• Purified, detergent-solubilized MPs
• APol, usually A8-35, but we have also investigated the utility of A8-75, A34-35, A34-75,
SAPol, and NAPol (see Chap. 4) for ESI-IM-MS, with mixed results (Watkinson et al. 2015)
• Caesium iodide solution (2 g‧L
À1 ) in 50% (v/v) water/propan-2-ol
• IM calibrants (cytochrome c, bovine serum albumin, concanavalin A, alcohol dehydrogenase)
• ESI-MS compatible buffers: ammonium acetate and ammonium bicarbonate
• Buffer-exchange devices (e.g. desalting columns, dialysis apparatus)
• Gold/palladium-coated nano-ESI needles
• GELoader tips (Eppendorf)
• Synapt HDMS mass spectrometer, equipped with static nanoflow-ESI (nano-ESI) source
(Waters Corporation)
648
14 The Use of Amphipols in Mass Spectrometry
Proteins by Electrospray Ionization-Ion Mobility Spectrometry-Mass
Spectrometry
14.5.1 Introduction
Non-covalent (or “native”) mass spectrometry (MS) is emerging as an invaluable method to study
membrane proteins (MPs) and MP complexes, as well as their interactions with lipids and small
molecules (Laganowsky et al. 2014; Bechara et al. 2015; Bechara and Robinson 2015; Konijnenberg
et al. 2015; Rajabi et al. 2015; Gault et al. 2016). The approach requires samples to be maintained in
their native conformations in solution before ionization, typically by the use of electrospray ionization
(ESI). Implementation of non-covalent ESI-MS with ion mobility (IM) spectrometry, i.e. ESI-IM-MS,
allows measurement of the ions’ rotationally averaged collisional cross sections (CCSs), together with
their mass-to-charge (m/z) ratios, in a single experiment. Ion CCSs are related to shape, size, and
charge, and can yield structural information about biomolecules (Ruotolo et al. 2008).
Typically, MP analysis using ESI-IM-MS has been performed by solubilizing the protein of
interest with detergents and subsequently dissociating the ionized MP/detergent complex in vacuo by
collisional activation, leading to the ejection of the MP from its solubilizing media for mass analysis
(Barrera et al. 2008; Laganowsky et al. 2013). This is key to successful MS analyses, as MP/surfactant
complexes are highly heterogeneous, and therefore measurement of a complex’s intact mass is not
possible. We, and others, have developed detergent-free methods for the study of MPs using ESI-MS,
by introducing MPs solubilized with amphipols (APols), bicelles, or nanodiscs into the gas phase for
interrogation (Hopper et al. 2013; Calabrese et al. 2015; Watkinson et al. 2015; Marty et al. 2016a).
Here, a general approach is described for the study of APol-solubilized MPs using ESI-IM-MS,
highlighting key stages of the sample preparation and MS analyses that must be optimized for each
system.
In the following, the protocol itself is printed in roman, background and comments in italics,
preceded by a pointing hand ().
14.5.2 Materials
• Purified, detergent-solubilized MPs
• APol, usually A8-35, but we have also investigated the utility of A8-75, A34-35, A34-75,
SAPol, and NAPol (see Chap. 4) for ESI-IM-MS, with mixed results (Watkinson et al. 2015)
• Caesium iodide solution (2 g‧L
À1 ) in 50% (v/v) water/propan-2-ol
• IM calibrants (cytochrome c, bovine serum albumin, concanavalin A, alcohol dehydrogenase)
• ESI-MS compatible buffers: ammonium acetate and ammonium bicarbonate
• Buffer-exchange devices (e.g. desalting columns, dialysis apparatus)
• Gold/palladium-coated nano-ESI needles
• GELoader tips (Eppendorf)
• Synapt HDMS mass spectrometer, equipped with static nanoflow-ESI (nano-ESI) source
(Waters Corporation)
648
14 The Use of Amphipols in Mass Spectrometry