Hydrogen/deuterium exchange on yeast ATPase supramolecular protein complex analyzed at high sensitivity by MALDI mass spectrometry.
To evaluate the ability of hydrogen/deuterium exchange of amide protons followed by mass spectrometry (HXMS) to yield topological information about supramolecular protein complexes, this approach has been tested with the 370 kDa hetero-oligomeric complex of yeast F1-ATPase. The study was focused on the epsilon subunit (6612 Da) of the complex. Deuterium back exchange due to the chromatographic isolation step of this subunit was strongly reduced by means of fast micro-chromatography, and MALDI-MS was used to analyze either the intact subunit or peptide mixtures resulting from its proteolytic cleavage. A deuterium labeling kinetic study was conducted with epsilon subunit being a part of the F1 native complex. The effect of a secondary structure was also investigated by means of HXMS on the isolated epsilon subunit. Finally, to determine which regions of epsilon subunit are accessible to solvent in F1-ATPase during exchange, the complex was submitted to hydrogen/deuterium exchange, the epsilon subunit was purified by micro-chromatography, digested by pepsin, and resulting peptide fragments were analyzed by MALDI-MS. The combination of hydrogen/deuterium exchange, fast micro-chromatography and MALDI-MS was shown to be a fast and efficient way to obtain detailed topological information for the epsilon subunit when it is engaged in the ATPase complex.
Nazabal
Alexis
A
Institut Européen de Chimie et Biologie, Pessac, France. a.nazabal@iecb-polytechnique.u.bordeaux.fr
Laguerre
Michel
M
Schmitter
Jean-Marie
JM
Vaillier
Jacques
J
Chaignepain
Stéphane
S
Velours
Jean
J
D000595:Amino Acid Sequence; D003903:Deuterium / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D007474:Ion Exchange; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D015003:Yeasts / Q000201:enzymology*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D021122:Protein Subunits; D006859:Hydrogen; D003903:Deuterium; D010434:Pepsin A; D006180:Proton-Translocating ATPases