Isolation and Quantitation of a Minor Determinant of Hen Egg White Lysozyme Bound to I-A super(k) by Using Peptide-Specific Immunoaffinity

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-A super(k) of APCs. We isolated and concentrated the peptides from the I-A super(k) extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34-45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33-45), and the longest peptide was a 19-mer (residues 31-49). The two most abundant were 31-47 (1.3 pmol) and 31-46 (1 pmol), while the least abundant were 31-45 (40 fmol) and 32-45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31-47 peptide, the predominant member of this series, was 22 times lower than that of 48-62, the major epitope of HEL. The 31-47 peptide bound about 20-fold weaker to I-A super(k) compared with the dominant 48-62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.