Fan Chen1, Sabina Gerber2, Katrin Heuser2, Vladimir M. Korkhov2, Christian Lizak2, Samantha Mireku2, Kaspar P. Locher2, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland
- Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerlan
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.
CovalX Technology Used (Click each option to learn more)
PglB, PglK, BtuCD, BtuCDF, and Cdr1p were purified, respectively. The protein complexes were cross-linked by mixing them with 10% glutaraldehyde (GA) solution in a 10/1 (v/v) ratio for a predetermined amount of time at room temperature in buffer solutions that contained detergent micelles. The samples were mixed with the matrix (sinapic acid (20 mg/mL in water/acetonitrile/TFA, 49.95/49.95/0.1, v/v/v) in a ½ (v/v) ratio. 1 μl of the mixture was spotted on a stainless steel MALDI plate and dried under ambient conditions. The samples were analyzed using a mass spectrometer that had been modified with a CovalX HM2 detection system. From this analysis, researchers were able to determine that integral membrane proteins can be detected and their MW can be determined with high mass accuracy. The ability of MALDI-MS to provide stoichiometry of complexes and post-translational modifications allows for the method to become a tool for membrane protein drug target discovery and study.