• APol-trapped MPs were then dialyzed against 100 mM ammonium bicarbonate, pH 8.0, at
4
C for 24 h.
횿Ammonium bicarbonate was chosen as the buffer, because below pH 7 MP/APol
complexes aggregate (see Chap. 4, § 4.3.1.2.2). The optimal buffering range for ammonium
bicarbonate is pH 8.2–11.3. Ammonium acetate, the other commonly used MS-compatible
buffer, is most efficient as a buffer at pH 3.8–5.8, and hence not suitable for analyses of MP/
A8-35 complexes.
It may be necessary to perform size exclusion chromatography (SEC) of APol-trapped MPs
prior to ESI-IM-MS analysis. SEC enables removal of free APol, which, in our experience,
enhances the detectability of MPs. For example, when the transmembrane β-barrel domain of
OmpA (tOmpA) was trapped in A34-35, a mass spectrum could only be acquired after SEC
(Watkinson et al. 2015).
• Preparation of IM calibrants: cytochrome c, bovine serum albumin, concanavalin A, and
alcohol dehydrogenase (Bush et al. 2010) were solubilized in 200 mM ammonium acetate
solution (concentration ~10 μM) and desalted using spin desalting columns (e.g. Micro
BioSpin 6, Bio-Rad) according to the manufacturer’s instructions.
2. ESI-IM-MS analysis
Several IM-enabled mass spectrometers are commercially available. Our analyses were
performed on a Waters Synapt HDMS instrument, a hybrid quadrupole-travelling wave ion
mobility-time-of-flight mass spectrometer (Fig. 14.17) (Pringle et al. 2007). The travelling
wave (T-wave) sector of the instrument comprises an IM cell, sandwiched between two
collision cells (termed the “trap” and “transfer” cells). Our instrument is fitted with a static
nano-ESI source, and a quadrupole capable of mass selection up to m/z ¼ 32,000 (Sobott
et al. 2002). For nano-ESI, samples are ionized by loading into an electrically conductive
capillary, which is placed near the instrument’s sampling cone. An electric potential is
applied to the capillary, resulting in a Taylor cone, and the formation of ions which are
drawn into the mass spectrometer as a result of the potential difference between the capillary
and sampling cone.
• Capillaries are commercially available (e.g. from Waters Corporation and Proxeon
Biosystems) or can be prepared in-house. We use borosilicate glass capillaries that are pulled
to a tapered tip using a micropipette puller (Model P-97, Sutter Instruments) and coated using
a sputter coater fitted with a gold/palladium target (Emitech Sc7620). Capillary tips are
trimmed (with tweezers, the capillary being visualized with a light microscope) before use
to obtain a stable spray. The cut capillary has often an internal diameter of 1–10 μm (this is a
trial and error process) (Hernandez and Robinson 2007).
For evidence that ionization by nano-ESI leads to native protein conformations and
interactions being retained in vacuo see e.g. Ruotolo et al. (2005), Ruotolo and Robinson
(2006), and Hilton and Benesch (2012).
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14 The Use of Amphipols in Mass Spectrometry
4
C for 24 h.
횿Ammonium bicarbonate was chosen as the buffer, because below pH 7 MP/APol
complexes aggregate (see Chap. 4, § 4.3.1.2.2). The optimal buffering range for ammonium
bicarbonate is pH 8.2–11.3. Ammonium acetate, the other commonly used MS-compatible
buffer, is most efficient as a buffer at pH 3.8–5.8, and hence not suitable for analyses of MP/
A8-35 complexes.
It may be necessary to perform size exclusion chromatography (SEC) of APol-trapped MPs
prior to ESI-IM-MS analysis. SEC enables removal of free APol, which, in our experience,
enhances the detectability of MPs. For example, when the transmembrane β-barrel domain of
OmpA (tOmpA) was trapped in A34-35, a mass spectrum could only be acquired after SEC
(Watkinson et al. 2015).
• Preparation of IM calibrants: cytochrome c, bovine serum albumin, concanavalin A, and
alcohol dehydrogenase (Bush et al. 2010) were solubilized in 200 mM ammonium acetate
solution (concentration ~10 μM) and desalted using spin desalting columns (e.g. Micro
BioSpin 6, Bio-Rad) according to the manufacturer’s instructions.
2. ESI-IM-MS analysis
Several IM-enabled mass spectrometers are commercially available. Our analyses were
performed on a Waters Synapt HDMS instrument, a hybrid quadrupole-travelling wave ion
mobility-time-of-flight mass spectrometer (Fig. 14.17) (Pringle et al. 2007). The travelling
wave (T-wave) sector of the instrument comprises an IM cell, sandwiched between two
collision cells (termed the “trap” and “transfer” cells). Our instrument is fitted with a static
nano-ESI source, and a quadrupole capable of mass selection up to m/z ¼ 32,000 (Sobott
et al. 2002). For nano-ESI, samples are ionized by loading into an electrically conductive
capillary, which is placed near the instrument’s sampling cone. An electric potential is
applied to the capillary, resulting in a Taylor cone, and the formation of ions which are
drawn into the mass spectrometer as a result of the potential difference between the capillary
and sampling cone.
• Capillaries are commercially available (e.g. from Waters Corporation and Proxeon
Biosystems) or can be prepared in-house. We use borosilicate glass capillaries that are pulled
to a tapered tip using a micropipette puller (Model P-97, Sutter Instruments) and coated using
a sputter coater fitted with a gold/palladium target (Emitech Sc7620). Capillary tips are
trimmed (with tweezers, the capillary being visualized with a light microscope) before use
to obtain a stable spray. The cut capillary has often an internal diameter of 1–10 μm (this is a
trial and error process) (Hernandez and Robinson 2007).
For evidence that ionization by nano-ESI leads to native protein conformations and
interactions being retained in vacuo see e.g. Ruotolo et al. (2005), Ruotolo and Robinson
(2006), and Hilton and Benesch (2012).
650
14 The Use of Amphipols in Mass Spectrometry