Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features.
The crystallization and structure determination of integral membrane proteins remains a difficult task relying on a good understanding of the behavior of the protein for success. To date, membrane protein structures are still far outnumbered by soluble protein structures. Mass spectrometry is a powerful and versatile tool offering deep insights into the state of the integral membrane protein the structuralist intends to crystallize. With appropriate sample preparation methods, it provides information that can sometimes prove critical at various stages of the structure determination process, from protein expression to model building. Moreover, valuable knowledge is gained when the identified structural features underlie important functional aspects. Electrospray and matrix assisted laser desorption ionization (MALDI) methods, however, face a particular challenge when dealing with integral membrane proteins. A MALDI method specifically optimized for membrane protein analysis is presented here, with detailed information on the sample preparation and deposition, as well as guidelines for domain determination by limited proteolysis. MALDI-time of flight mass spectrometry can be used to do a proper inventory of initiation sites, to tailor a protein to a stable, well-folded form, and to evaluate selenomethionine replacement. These approaches are illustrated with a few examples drawn from the structural biology of ion channels.
Gabant
Guillaume
G
Centre de Biophysique Moléculaire, CNRS UPR4301, Rue Charles Sadron, 45071 Orleans cedex 2, France.
Cadene
Martine
M
D003460:Crystallization; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012645:Selenomethionine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D007473:Ion Channels; D008565:Membrane Proteins; D012645:Selenomethionine; D010447:Peptide Hydrolases