Author: Mundt A.A. Cuillel M. Forest E. Dupont Y.
Publisher: Elsevier
ISSN: 0003-2697
Source: Analytical Biochemistry, Vol.299, Iss.2, 2001-12, pp. : 147-157
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Abstract
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 Ca2+-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) Ca2+-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 Ca2+-ATPase.
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