Comparative peptide mapping of a hepatitis C viral recombinant protein by capillary electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry

• Published in: Journal of Chromatography A Vol. 744; no. 1; pp. 177 - 185
• Main Authors: Winkler, Martin A., Kundu, Samar, Robey, Thomas E., Gerard Robey, W.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 13.09.1996
• Subjects: Alkylation; Amino Acid Sequence; Antigens, Viral - analysis; Antigens, Viral - chemistry; Antigens, Viral - metabolism; Electrophoresis, Capillary - methods; Hepacivirus - immunology; Hepacivirus - metabolism; Hepatitis C virus antigen; Metalloendopeptidases - metabolism; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments - analysis; Peptide Fragments - chemistry; Peptide Mapping; Peptides; Proteins; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reproducibility of Results; Sodium Dodecyl Sulfate - chemistry; Software; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Proteins; Peptides; Peptide mapping; Hepatitis C virus antigen
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(96)00184-7

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.