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 d ifferentiation factor EGF domain in order to obtain improved peptide maps u seful for assignment of disulfide linkages. The procedure described here in volves an on-line digestion of proteins using immobilized trypsin and endop roteinase Glu-C cartridges connected in series, followed by on-line RP-HPLC separation of the peptides. The entire process can be automated using a co mmercially available workstation; and the total time required for both prot eolytic digestion and the HPLC separation can be shortened to within 1 h. U sing these immobilized columns, we demonstrated that disulfide structure as signment 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 p eptides using matrix-assisted laser desorption/ionization time-of-flight ma ss spectrometry. The use of immobilized protease cartridges in tandem elimi nates 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. (C) 2000 Elsevier Science B.V. All rights reserved.