title: Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry.
abstract: Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.
authors.affiliation: Institute of Chemistry, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan, ROC.
authors.forename: Chih-Yang
authors.initials: CY
authors.lastname: Chang
authors.forename: Hsin-Kai
authors.initials: HK
authors.lastname: Liao
authors.forename: Chiun-Gung
authors.initials: CG
authors.lastname: Juo
authors.forename: Shu-Hua
authors.initials: SH
authors.lastname: Chen
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen