MALDI-TOF mass spectrometry analysis of amphipol-trapped membrane proteins.
Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of  Escherichia coli  outer membrane protein A, a β-barrel, eubacterial MP, (ii)  Halobacterium salinarum  bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga  Chlamydomonas reinhardtii  and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.
Université Pierre et Marie Curie (UPMC Université Paris 06), Laboratoire des BioMolécules (LBM), Paris, France.
Chérine
C
Bechara
Gérard
G
Bolbach
Paola
P
Bazzaco
K Shivaji
KS
Sharma
Grégory
G
Durand
Jean-Luc
JL
Popot
Francesca
F
Zito
Sandrine
S
Sagan
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003902:Detergents / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D055767:Immobilized Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D003902:Detergents; D055767:Immobilized Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011108:Polymers; D014357:Trypsin