Peptide mapping and disulfide bond analysis of the cytoplasmic region of an intrinsic membrane protein by mass spectrometry.
Intrinsic membrane proteins pose substantial obstacles to analysis by common analytical techniques due to their hydrophobic nature and solubilization requirements. This is the case for studies involving HPLC coupled to mass spectrometry. We have developed an HPLC/mass spectrometry approach to explore and map the peptide sequence of the SERCA1a Ca(2+)-ATPase from the sarcoplasmic reticulum an integral membrane protein of 110 kDa. After extensive proteolysis of the protein, the mass of the proteolytic fragments was analyzed by HPLC/mass spectrometry. Only part of the cytoplasmic fragments was recovered under nondenaturing conditions. On the other hand, peptide fragments obtained under denaturing conditions were found to cover nearly all the cytoplasmic region. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase contains 24 cysteine residues, 18 of which are in the cytosolic or lumenal region of the protein. Peptides containing free cysteines were identified by a mass increase resulting from carboxyamidomethylation of the cysteines with iodoacetamide. Alkylation reactions were executed either before or after reduction of the peptide fragments by dithiothreitol. Analysis of the mass of the fragments indicates that no disulfide bonds exist in the cytoplasmic portion of SR Ca(2+)-ATPase.
Mundt
A A
AA
UMR-CNRS-CEA-UJF-5090, CEA, BMC-Département de Biologie Moléculaire et Structurale, CEA, 30854 Grenoble, France.
Cuillel
M
M
Forest
E
E
Dupont
Y
Y
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004220:Disulfides / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D011489:Protein Denaturation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D003902:Detergents; D004220:Disulfides; D008565:Membrane Proteins; D010455:Peptides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003545:Cysteine