DIFFUSIVE TRANSFER TO MEMBRANES AS AN EFFECTIVE INTERFACE BETWEEN GEL-ELECTROPHORESIS AND MASS-SPECTROMETRY

Diffusive transfer was examined as a blotting method to transfer prote ins from polyacrylamide gels to membranes for ultraviolet matrix-assis ted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spe ctra have been obtained for 11 pmol of 66 kDa albumin loaded onto an I EF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclona l antibody anti-beta-galactosidase transferred to Goretex. Polypropyle ne, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF req uired that the membrane be kept wet prior to application of matrix, Co nsiderations in mass accuracy for analysis from large-area membranes w ith continuous extraction and delayed extraction were explored, as wer e remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping. (C) 1997 Elsevier Scie nce B.V.