Dealing with intractable protein cores: protein sequencing of the Mcg IgG and the Yvo IgM heavy chain variable domains

Abstract The VH domains of two human monoclonal antibodies, designated Mcg IgG1(λ) and Yvo IgM(κ), were particularly intractable to standard protein sequencing protocols. Peptides liberated from the VH domains of these proteins, using standard enzymatic or chemical cleavages, invariably precipitated during the procedures. Boiling in SDS containing buffers dissolved precipitates and the peptides were separated using SDS–PAGE. Fully overlapped VH sequences were obtained with a series of ‘in‐gel’ cleavages, followed by passive/differential transfers of peptides onto PVDF membranes. Both the in‐gel cleavages and passive transfers could be applied to ‘wet’ or ‘dry’ gels so that gels could be archived and used at a later date to obtain additional sequence information from a fragment of interest. Repetitive yields of even the most insoluble peptides were such that the sequences of various peptides from relatively complex mixtures of peptides could be assigned with confidence. Despite the overall success of the sequencing, we occasionally referred to electron density maps, calculated for crystals of the Fab of Yvo IgM, to resolve particular sequences and confirm ambiguous amino acid assignments. Methods we describe in this report should be generally useful for obtaining sequences of proteins with intractable cores and may find many applications in the ‘post genomic era’. Copyright © 2002 John Wiley & Sons, Ltd.