Analysis of recombinant Schistosoma mansoni antigen rSmp28 by on-line liquid chromatography-mass spectrometry combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.
A recombinant Schistosoma mansoni antigen produced in Saccharomyces cerevisiae and purified by glutathione-Sepharose affinity chromatography was analyzed by tryptic peptide mapping using on-line reversed-phase high-performance liquid chromatography pneumatically assisted electrospray mass spectrometry confirming the complete primary structure. Partial covalent modification of the single cysteine in the protein with glutathione as well as partial dimerization of the Cys-containing tryptic peptide was observed. Combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and tryptic digestion of the monomeric protein in the gel slice revealed that dimerization was occurring during enzymatic digestion. Furthermore, part of the Cys-containing fragment was covalently modified with one moiety of beta-mercaptoethanol by the electrophoresis sample buffer and five of the seven methionine-containing peptide fragments were partially oxidized to the respective sulfoxides. The use of capillary columns provided a complete peptide map of rSmp28 on 7 pmol of tryptic digest after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Klarskov
K
K
Université Louis Pasteur, Laboratoire de Spectrométrie de Masse Bioorganique, URA 31, CNRS, Strasbourg, France.
Roecklin
D
D
Bouchon
B
B
Sabatié
J
J
Van Dorsselaer
A
A
Bischoff
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015801:Helminth Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis; D012550:Schistosoma mansoni / Q000276:immunology*; D012967:Sodium Dodecyl Sulfate
D000947:Antigens, Helminth; D015801:Helminth Proteins; C052358:P28 antigen, Schistosoma; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate