surfactant either DDM or any of six different APols. The average mass (hM n i) and percentage of
charged units in each APol are given in the left column of Fig. 14.11. As described in Chap. 4, § 4.2.1,
A34-35 has the same chemical structure and charge density as A8-35, but its molecules are, on
average, ~4Â longer. A8-75 has the same length distribution as A8-35, but it has not been grafted
with isopropylamine, and, therefore, it retains ~75% rather than ~35% of free carboxylates. A34-75 is
a ~4Â longer version of A8-75. These four APols were initially described in Tribet et al. 1996. The
sulfonated APol (SAPol) is a version of A8-75 that has been grafted with ~40% taurine (Dahmane et al.
2011) (Chap. 4, § 4.2.2.1, Fig. 4.1). Its charge density and average length are the same as A8-75, but,
because it carries sulfonates, its solubility is insensitive to the pH and to the presence of divalent
cations. The last APol tested is a glucosylated, homotelomeric, non-ionic APol (“NAPol”; see Chap. 4,
§ 4.2.3.2, Fig. 4.5) (Bazzacco et al. 2012; Sharma et al. 2012). Taken together, these six APols provide
an opportunity to test the effect of APol charge density and length on the transfer of MPs to the gas
phase, and the use of three MPs gives a chance to assess the generality of the conclusions (Fig. 14.11).
The first conclusion from this analysis is the idiosyncrasy of OMPs. Despite their structural
similarities (Fig. 14.11, top), they do not necessarily behave in the same way: tOmpA is relatively
indifferent to the surfactant it is ejected from, whereas OmpT and PagP are more picky about it,
meaning that they separate less easily without denaturation. The second point is that the chemical
structure of the APol does influence the efficiency of the release of protein ions carrying a minimum
amount of charges, which correlates with minimal denaturation (Watkinson et al. 2015): a longer and
more densely charged APol is less favorable than a shorter and less charged one. Nevertheless, a short,
totally uncharged APol (NAPol) does not give excellent results, the best results overall (on this
restricted set of MPs) being obtained with A8-35. These observations will be further discussed
below (§ 14.3.4). They are patchy, but they seem to be consistent with the view that the less polar
interactions there are between MP-associated APols, the higher the probability that they will desorb
without the protein denaturating, which may point the way to designing APols specifically optimized
for MS.
A detailed protocol for ESI-MS-IM examination of MP/APol complexes is given in § 14.5.
14.3.2 Amphipols, ESI-MS, and Oligomeric Membrane Proteins
Very little data are available about using APols to examine oligomeric MPs by ESI-MS. In Study 14.4,
a comparison was made of the release of MPs from detergents (DDM and n-decyl-β-D-maltopyranoside), APols (A8-35), nanodiscs, and bicelles (Hopper et al. 2013). The MPs studied were
E. coli lactose permease, a monomeric MP, E. coli diacylglycerol kinase (DAGK), a trimeric one, and
sensory rhodopsin II (pSRII) from Natronomonas pharaonis, a monomeric 7-TM-helix photoreceptor.
Only DAGK was studied as a MP/APol complex. As regards the identification of the correct
oligomeric state, only bicelles performed reasonably well, releasing a mixture of DAGK trimers,
monomers, and some dimers (Fig. 14.12C). DAGK released from A8-35 was mostly monomeric, with
some trimers and occasional dimers (Fig. 14.12B), and DAGK released from DDM a mixture of
monomers and dimers (Fig. 14.12A). Nanodiscs released a mixture of monomers, dimers, and trimers
(Hopper et al. 2013). Whether the inability of A8-35 to release DAGK specifically as the native,
physiological oligomer is specific to this protein, to A8-35, and/or to the conditions explored remains
to be examined. As discussed in § 14.3.4, the use of other APols, in particular, might be worth
considering.
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14 The Use of Amphipols in Mass Spectrometry
charged units in each APol are given in the left column of Fig. 14.11. As described in Chap. 4, § 4.2.1,
A34-35 has the same chemical structure and charge density as A8-35, but its molecules are, on
average, ~4Â longer. A8-75 has the same length distribution as A8-35, but it has not been grafted
with isopropylamine, and, therefore, it retains ~75% rather than ~35% of free carboxylates. A34-75 is
a ~4Â longer version of A8-75. These four APols were initially described in Tribet et al. 1996. The
sulfonated APol (SAPol) is a version of A8-75 that has been grafted with ~40% taurine (Dahmane et al.
2011) (Chap. 4, § 4.2.2.1, Fig. 4.1). Its charge density and average length are the same as A8-75, but,
because it carries sulfonates, its solubility is insensitive to the pH and to the presence of divalent
cations. The last APol tested is a glucosylated, homotelomeric, non-ionic APol (“NAPol”; see Chap. 4,
§ 4.2.3.2, Fig. 4.5) (Bazzacco et al. 2012; Sharma et al. 2012). Taken together, these six APols provide
an opportunity to test the effect of APol charge density and length on the transfer of MPs to the gas
phase, and the use of three MPs gives a chance to assess the generality of the conclusions (Fig. 14.11).
The first conclusion from this analysis is the idiosyncrasy of OMPs. Despite their structural
similarities (Fig. 14.11, top), they do not necessarily behave in the same way: tOmpA is relatively
indifferent to the surfactant it is ejected from, whereas OmpT and PagP are more picky about it,
meaning that they separate less easily without denaturation. The second point is that the chemical
structure of the APol does influence the efficiency of the release of protein ions carrying a minimum
amount of charges, which correlates with minimal denaturation (Watkinson et al. 2015): a longer and
more densely charged APol is less favorable than a shorter and less charged one. Nevertheless, a short,
totally uncharged APol (NAPol) does not give excellent results, the best results overall (on this
restricted set of MPs) being obtained with A8-35. These observations will be further discussed
below (§ 14.3.4). They are patchy, but they seem to be consistent with the view that the less polar
interactions there are between MP-associated APols, the higher the probability that they will desorb
without the protein denaturating, which may point the way to designing APols specifically optimized
for MS.
A detailed protocol for ESI-MS-IM examination of MP/APol complexes is given in § 14.5.
14.3.2 Amphipols, ESI-MS, and Oligomeric Membrane Proteins
Very little data are available about using APols to examine oligomeric MPs by ESI-MS. In Study 14.4,
a comparison was made of the release of MPs from detergents (DDM and n-decyl-β-D-maltopyranoside), APols (A8-35), nanodiscs, and bicelles (Hopper et al. 2013). The MPs studied were
E. coli lactose permease, a monomeric MP, E. coli diacylglycerol kinase (DAGK), a trimeric one, and
sensory rhodopsin II (pSRII) from Natronomonas pharaonis, a monomeric 7-TM-helix photoreceptor.
Only DAGK was studied as a MP/APol complex. As regards the identification of the correct
oligomeric state, only bicelles performed reasonably well, releasing a mixture of DAGK trimers,
monomers, and some dimers (Fig. 14.12C). DAGK released from A8-35 was mostly monomeric, with
some trimers and occasional dimers (Fig. 14.12B), and DAGK released from DDM a mixture of
monomers and dimers (Fig. 14.12A). Nanodiscs released a mixture of monomers, dimers, and trimers
(Hopper et al. 2013). Whether the inability of A8-35 to release DAGK specifically as the native,
physiological oligomer is specific to this protein, to A8-35, and/or to the conditions explored remains
to be examined. As discussed in § 14.3.4, the use of other APols, in particular, might be worth
considering.
640
14 The Use of Amphipols in Mass Spectrometry