High-Mass Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry of Integral Membrane Proteins and Their Complexes

详细信息    查看全文

• 作者：Fan Chen ; Sabina Gerber ; Katrin Heuser ; Vladimir M. Korkhov ; Christian Lizak ; Samantha Mireku ; Kaspar P. Locher ; Renato Zenobi
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：April 2, 2013
• 年：2013
• 卷：85
• 期：7
• 页码：3483-3488
• 全文大小：376K
• 年卷期：v.85,no.7(April 2, 2013)
• ISSN：1520-6882

文摘

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 鈮?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.