Structural characterization of recombinant hepatitis B surface antigen protein by mass spectrometry

Abstract

The primary structure of recombinant hepatitis B surface antigen protein produced in yeast has been confirmed by mass spectrometric peptide mapping. These studies corroborate more than 85% of the amino acid sequence derived by sequencing of the gene and identified the presence of an acetyl moiety on approximately 70% of the NH2-terminal methionine residues. Prior to the present work, direct structural analysis was largely prevented by the insolubility of this integral membrane protein and its primary degradation fragments in aqueous buffers and by partial blockage of the NH2 terminus. These difficulties were overcome by preparative isolation using electroelution of the monomeric 226 amino acid protein from a polyacrylamide electrophoretic gel in the presence of sodium dodecyl sulfate. Chymotryptic digestion of the reduced and carboxymethylated monomer produced a large number of small, predominantly hydrophobic peptides ideally suited for peptide mapping by fast atom bombardment mass spectrometry. The percentage of NH2-terminal methionine blocked by acetyl was determined by a new strategy involving cyanogen bromide cleavage, permethylation, and gas chromatography/mass spectrometry identification and quantitation of the N-methyl-N-acetylhomoserine produced.

Acridine- and cholesterol-derivatized solid supports for improved synthesis of 3'-modified oligonucleotides

“…The 3'-tailed 11-mer ODNs were prepared with a sequence complementary to the initiation codon region of the mRNA for the hepatitis B surface antigen protein (33). The 20-mer ODN target chosen for the Tm studies provides nucleotide overhangs that may influence interactions with the 3'-modification.…”

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

“…Manual and Automated Edman Degradation. Manual Edman degradation was performed on mixtures of peptides as previously described (Hemling et al, 1988). Automated Edman degradation was carried out with a Beckman System 890 M-2 sequencer with HPLC identification and quantitation of PTH-amino acids performed as previously described (Hawke et al, 1982).…”

Recombinant Virus-like Particle Protein Vaccines

“…Original data published by GSK (Stephenne 1990) indicated consistent levels of lipid (11-15 mcg per 20 mcg protein), sugars (0.2-0.35 mcg per 20 mcg protein), antigen using a radioimmunoassay called AUSRIA (Abbott_Diagnostics 2012), and protein purity (>98 %) in the bulk product. Further structural studies of the vaccine (Hemling et al 1988) confirmed 85 % of the sequence by FAB mass spectrometry of proteolytic and CNBr digests. Complete structure confirmation was thwarted by the fact that the protein was 70 % blocked at its N-terminus and contained very hydrophobic sequences making handling of the protein difficult.…”