title: High-mass matrix-assisted laser desorption ionization-mass spectrometry of integral membrane proteins and their complexes.
abstract: Analyzing purified membrane proteins and membrane protein complexes by mass spectrometry has been notoriously challenging and required highly specialized buffer conditions, sample preparation methods, and apparatus. Here we show that a standard matrix-assisted laser desorption/ionization (MALDI) protocol, if used in combination with a high-mass detector, allows straightforward mass spectrometric measurements of integral membrane proteins and their complexes, directly following purification in detergent solution. Molecular weights can be determined precisely (mass error ≤ 0.1%) such that high-mass MALDI-MS was able to identify the site for N-linked glycosylation of the eukaryotic multidrug ABC transporter Cdr1p without special purification steps, which is impossible by any other current approach. After chemical cross-linking with glutaraldehyde in the presence of detergent micelles, the subunit stoichiometries of a series of integral membrane protein complexes, including the homomeric PglK and the heteromeric BtuCD as well as BtuCDF, were unambiguously resolved. This thus adds a valuable tool for biophysical characterization of integral membrane proteins.
authors: Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, Switzerland.
authors: Fan
authors: F
authors: Chen
authors: Sabina
authors: S
authors: Gerber
authors: Katrin
authors: K
authors: Heuser
authors: Vladimir M
authors: VM
authors: Korkhov
authors: Christian
authors: C
authors: Lizak
authors: Samantha
authors: S
authors: Mireku
authors: Kaspar P
authors: KP
authors: Locher
authors: Renato
authors: R
authors: Zenobi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002167:Campylobacter / Q000737:chemistry; D002176:Candida albicans / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins