Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, the use of DEL as a model protein has been complicated by the presence of multiple isoforms and conflicting reports of primary sequence.
In this study, three isoforms of DEL (DEL-I to DEL-III) were purified from Pekin duck (Anas platyrhynchos) eggs by carboxymethyl ion-exchange chromatography. In parallel with crystallography studies, the fractions were assessed by SDS-PAGE analysis and the three isoforms were shown to be well separated. The accurate intact mass of each the isoform was obtained by direct infusion on an Orbitrap Fusion mass spectrometer. Complementary sets of peptides were generated by proteolytic digestion with trypsin or Lys-C proteases. The peptides were then analysed either by MALDI TOF MS, using a QSTAR Elite QTOF mass spectrometer, or by Nanospray QTOF MS, using a 6600 QTOF mass spectrometer. By utilising multiple mass spectrometry approaches in conjunction with crystallographic data it was possible to report the non-ambiguous primary sequences for DEL-I to DEL-III.
Analysis of the primary sequences explains the sequential elution of the isoforms from the carboxymethyl resin. We were also able to resolve the identity of residues 66 and 103 which have been consistently problematic in their assignment in previously published reports. With the use of a protein-fold ‘all-on-all’ dendogram, it was now possible to relate the duck lysozymes to those found in other fowl, mammals, and animals.