Universal isolation of cross-linked peptides: Application to neurofibrillary tangles

A universal procedure to isolate cross-linked peptides has been demonstrate d. The procedure relies on initially converting the E-amino groups of lysin e to dimethyl lysine by reductive methylation with sodium cyanoborohydride and formaldehyde. The lysine-modified protein is proteolytically cleaved an d the resulting cl-amino groups derivatized with methoxypoly(ethylene glyco l)(5000) succinyl hydroxy-succinimide. Any unintentional derivatization of tyrosine side chains can be reversed by incubation under mildly alkaline co nditions. The cross-linked polypeptides contain two poly(ethylene glycol)(5 000) chains while non-cross-linked peptides contain a single poly(ethylene glycol)(5000) chain. The two populations of peptides can be separated by ge l filtration chromatography. This procedure has been shown capable of isola ting cross-linked peptides using glutathione, lysozyme, chemically cross-li nked hemoglobin, and neurofibrillary tangles isolated from the brain of a c ase of Alzheimer's disease.