Shinichi Hara

Peptide map procedure using immobilized protease cartridges in tandem for disulfide linkage identification of neu differentiation factor epidermal growth factor domain

Immobilized proteolytic enzyme cartridges were used to rapidly digest neu differentiation factor EGF domain in order to obtain improved peptide maps useful for assignment of disulfide linkages. The procedure described here involves an on-line digestion of proteins using immobilized trypsin and endoproteinase Glu-C cartridges connected in series, followed by on-line RP-HPLC separation of the peptides. The entire process can be automated using a commercially available workstation; and the total time required for both proteolytic digestion and the HPLC separation can be shortened to within 1 h. Using these immobilized columns, we demonstrated that disulfide structure assignment of the EGF domains of recombinant human neu differentiation factor can be performed by isolation of individual disulfide-containing peptides followed by assignment of disulfide linkages with prompt fragmentation of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The use of immobilized protease cartridges in tandem eliminates undesirable digestion artifacts associated with longer digestion time and higher protease-to-substrate ratio and results in the development of a reproducible and high quality peptide map.

Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.