Comparative peptide mapping of a hepatitis C viral recombinant protein by capillary electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry.
Capillary electrophoresis (CE) and matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated as alternatives to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis for peptide mapping with Staphylococcus aureus protease (V8) of a hydrophobic recombinant hepatitis C virus antigen, HC-31, which required 0.1% SDS for solubility. Controls (V8 only) or HC-31 digests were extracted with chloroform-methanol-water (1:4:3) to remove SDS, which interferes with MALDI-TOF, and high salt content, which affects CE. In two different runs by CE, the elution times of each of 11 peptide peaks were very reproducible (R.S.D. < 0.016). 25 fragments were resolved by MALDI-TOF-MS, including six smaller peptides (M(r) < 13 000) resulting from V8 autodigestion. MALDI-TOF-MS indicated that partial cleavages occurred, primarily at sites where there are paired glutamic and/or aspartic acid residues.
Winkler
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Diagnostics Division, Abbott Laboratories, North Chicago, II 60064, USA.
Kundu
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Robey
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Robey
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WG
D000478:Alkylation; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D016174:Hepacivirus / Q000276:immunology* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000956:Antigens, Viral; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus