A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex.
A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.
Morgner
Nina
N
Institute for Physical and Theoretical Chemistry, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
Kleinschroth
Thomas
T
Barth
Hans-Dieter
HD
Ludwig
Bernd
B
Brutschy
Bernhard
B
D014450:Electron Transport Complex III / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008958:Models, Molecular; D010231:Paracoccus denitrificans / Q000737:chemistry* / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis* / Q000737:chemistry
D008565:Membrane Proteins; D021122:Protein Subunits; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III