In conclusion, Study 14.2 shows that MALDI-MS is applicable to APol-trapped MPs, with or
without trypsinolysis. It does not reveal more proteins nor more peptides than detergent-solubilized
preparations do, but it presents, nevertheless, a number of conceivable advantages. For one thing, the
mildness of APols should facilitate MS studies of fragile complexes and supercomplexes. In particular,
it may help identify subunits, cofactors, or lipids that are easily lost during MP purification in detergent
solution. More hypothetically, comparison of proteolytic fragments produced in a detergent vs. an
APol environment may provide hints as to which cleavage sites are naturally accessible and which
become so under the destabilizing effect of the detergent.
14.3 Electrospray Ionization-Mass Spectrometry Studies
ESI-MS studies were undertaken to answer the following questions: (i) Are MP/APol complexes
amenable to such studies, and what advantages and drawbacks do they present as compared to
MP/detergent ones? (ii) Which APols should preferably be used? (iii) Can such studies reveal
differences in the way detergents and APols interact with MPs?
14.3.1 Membrane Protein/Amphipol Complexes Are Amenable to Electrospray
Mass Spectrometry
The groups of Alison E. Ashcroft, Sheena E. Radford, and Peter J. F. Henderson have explored the
application of APols to ESI-MS, taking as models four monomeric bacterial MPs, two of them
featuring TM β-barrels, the other two TM α-helix bundles, whose structures are shown in Fig. 14.8.
Fig. 14.8 Three-dimensional structures and cellular localization of four bacterial membrane proteins used
to explore the amenability of membrane protein/amphipol complexes to electrospray mass spectrometry.
Crystal structures of PagP (PDB file 1THQ), OmpT (PDB file 1I78), and Mhp1 (PDB file 2X79) and a
homology model of GalP based on the crystal structure of XylE, which shares 30% sequence identity with
GalP (Adapted from Calabrese et al. 2015).
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14 The Use of Amphipols in Mass Spectrometry
without trypsinolysis. It does not reveal more proteins nor more peptides than detergent-solubilized
preparations do, but it presents, nevertheless, a number of conceivable advantages. For one thing, the
mildness of APols should facilitate MS studies of fragile complexes and supercomplexes. In particular,
it may help identify subunits, cofactors, or lipids that are easily lost during MP purification in detergent
solution. More hypothetically, comparison of proteolytic fragments produced in a detergent vs. an
APol environment may provide hints as to which cleavage sites are naturally accessible and which
become so under the destabilizing effect of the detergent.
14.3 Electrospray Ionization-Mass Spectrometry Studies
ESI-MS studies were undertaken to answer the following questions: (i) Are MP/APol complexes
amenable to such studies, and what advantages and drawbacks do they present as compared to
MP/detergent ones? (ii) Which APols should preferably be used? (iii) Can such studies reveal
differences in the way detergents and APols interact with MPs?
14.3.1 Membrane Protein/Amphipol Complexes Are Amenable to Electrospray
Mass Spectrometry
The groups of Alison E. Ashcroft, Sheena E. Radford, and Peter J. F. Henderson have explored the
application of APols to ESI-MS, taking as models four monomeric bacterial MPs, two of them
featuring TM β-barrels, the other two TM α-helix bundles, whose structures are shown in Fig. 14.8.
Fig. 14.8 Three-dimensional structures and cellular localization of four bacterial membrane proteins used
to explore the amenability of membrane protein/amphipol complexes to electrospray mass spectrometry.
Crystal structures of PagP (PDB file 1THQ), OmpT (PDB file 1I78), and Mhp1 (PDB file 2X79) and a
homology model of GalP based on the crystal structure of XylE, which shares 30% sequence identity with
GalP (Adapted from Calabrese et al. 2015).
636
14 The Use of Amphipols in Mass Spectrometry