14.5.3 Protocol
1. Preparation of samples for ESI-IM-MS
Conventional buffers used for biochemical analyses, such as >1 mM Tris/HCl or phosphate, are unsuitable for ESI-MS as they are nonvolatile and result in ion suppression and/or
adduct formation. Therefore, MPs (and all biomolecules undergoing native ESI-MS analysis)
must be transferred to a MS-compatible buffer before analysis. ESI-MS-compatible, volatile
buffers include ammonium acetate or ammonium bicarbonate, which are routinely used at
concentrations up to 1 M, depending on the desired pH and ionic strength (Hernandez and
Robinson 2007). Buffer exchange can be achieved by a number of means, including dialysis,
desalting columns, size exclusion chromatography, or ultrafiltration; however, it should be
ensured that stringent buffer exchange is employed, as nonvolatile buffer contaminants can
result in poor spectral quality. Nano-ESI is often used for non-covalent MS analyses, where
sample flow rates of a few nL‧min
À1 are used (compared with μL‧min
À1 for conventional
ESI), because the method is more sensitive than conventional ESI, is more tolerant to buffer
contaminants, and requires lower amounts of sample (~1 μL of sample can be used to obtain
a mass spectrum). Alternatively, matrix-assisted laser desorption ionization (MALDI) can be
used to ionize proteins for MS. APols have been shown to be compatible with MALDI-MS
(Bechara et al. 2012); however, ionization by this method does not permit native structure and
interactions to be maintained in vacuo.
Typically, we have used detergent-solubilized MPs for subsequent trapping in APols
(Calabrese et al. 2015). However, β-barrel outer membrane proteins refolded directly into
APols have also been used for ESI-MS analysis (Leney et al. 2012). For this work, a range of
APols has been screened. Among them, A8-35 showed the most promise as a generic
solubilizing agent, allowing ESI-IM-MS spectra to be recorded for the greatest range of
MPs tested (Watkinson et al. 2015).
In our study of both outer membrane and inner membrane proteins, the following sample
preparation procedure was performed:
• Detergent-solubilized MP samples were prepared in 10 mM Tris/HCl, pH 8.0, 0.02% (w/v)
DDM. For ESI-MS, protein concentrations in the low μM range are required. We routinely
prepare 50–500-μL samples (depending on protein availability) at protein concentrations
ca. 10 μM.
• APol was added to the detergent-solubilized membrane protein at a MP/APol ratio of 1:2 up
to 1:5 (w/w), and the samples were incubated on ice for 30 min.
In our experience, lower quantities of free APol in solution result in increased spectral
quality, so it is recommended that the amount of added APol be minimized for each sample,
without compromising sample integrity. This should be optimized empirically for each
protein by determining the lowest amount of APol to add without protein aggregation
occurring.
• Bio-Beads were prepared by washing with methanol (3Â), followed by water (3Â) and the
sample buffer (10 mM Tris/HCl, pH 8.0, with no added detergent). The pre-wet, washed,
Bio-Beads were added (20 g wet beads per g of detergent) to remove the detergent and the
sample mixture incubated for 1 h at 4
C with gentle agitation.
14.5 Protocol 14.1: Characterization of Amphipol-Trapped Membrane Proteins. . .
649
1. Preparation of samples for ESI-IM-MS
Conventional buffers used for biochemical analyses, such as >1 mM Tris/HCl or phosphate, are unsuitable for ESI-MS as they are nonvolatile and result in ion suppression and/or
adduct formation. Therefore, MPs (and all biomolecules undergoing native ESI-MS analysis)
must be transferred to a MS-compatible buffer before analysis. ESI-MS-compatible, volatile
buffers include ammonium acetate or ammonium bicarbonate, which are routinely used at
concentrations up to 1 M, depending on the desired pH and ionic strength (Hernandez and
Robinson 2007). Buffer exchange can be achieved by a number of means, including dialysis,
desalting columns, size exclusion chromatography, or ultrafiltration; however, it should be
ensured that stringent buffer exchange is employed, as nonvolatile buffer contaminants can
result in poor spectral quality. Nano-ESI is often used for non-covalent MS analyses, where
sample flow rates of a few nL‧min
À1 are used (compared with μL‧min
À1 for conventional
ESI), because the method is more sensitive than conventional ESI, is more tolerant to buffer
contaminants, and requires lower amounts of sample (~1 μL of sample can be used to obtain
a mass spectrum). Alternatively, matrix-assisted laser desorption ionization (MALDI) can be
used to ionize proteins for MS. APols have been shown to be compatible with MALDI-MS
(Bechara et al. 2012); however, ionization by this method does not permit native structure and
interactions to be maintained in vacuo.
Typically, we have used detergent-solubilized MPs for subsequent trapping in APols
(Calabrese et al. 2015). However, β-barrel outer membrane proteins refolded directly into
APols have also been used for ESI-MS analysis (Leney et al. 2012). For this work, a range of
APols has been screened. Among them, A8-35 showed the most promise as a generic
solubilizing agent, allowing ESI-IM-MS spectra to be recorded for the greatest range of
MPs tested (Watkinson et al. 2015).
In our study of both outer membrane and inner membrane proteins, the following sample
preparation procedure was performed:
• Detergent-solubilized MP samples were prepared in 10 mM Tris/HCl, pH 8.0, 0.02% (w/v)
DDM. For ESI-MS, protein concentrations in the low μM range are required. We routinely
prepare 50–500-μL samples (depending on protein availability) at protein concentrations
ca. 10 μM.
• APol was added to the detergent-solubilized membrane protein at a MP/APol ratio of 1:2 up
to 1:5 (w/w), and the samples were incubated on ice for 30 min.
In our experience, lower quantities of free APol in solution result in increased spectral
quality, so it is recommended that the amount of added APol be minimized for each sample,
without compromising sample integrity. This should be optimized empirically for each
protein by determining the lowest amount of APol to add without protein aggregation
occurring.
• Bio-Beads were prepared by washing with methanol (3Â), followed by water (3Â) and the
sample buffer (10 mM Tris/HCl, pH 8.0, with no added detergent). The pre-wet, washed,
Bio-Beads were added (20 g wet beads per g of detergent) to remove the detergent and the
sample mixture incubated for 1 h at 4
C with gentle agitation.
14.5 Protocol 14.1: Characterization of Amphipol-Trapped Membrane Proteins. . .
649