Universal Isolation of Cross-Linked Peptides:  Application to Neurofibrillary Tangles

A universal procedure to isolate cross-linked peptides has been demonstrated. The procedure relies on initially converting the epsilon-amino groups of lysine to dimethyl lysine by reductive methylation with sodium cyanoborohydride and formaldehyde. The lysine-modified protein is proteolytically cleaved and the resulting alpha-amino groups derivatized with methoxypoly(ethylene glycol)5000 succinyl hydroxysuccinimide. Any unintentional derivatization of tyrosine side chains can be reversed by incubation under mildly alkaline conditions. The cross-linked polypeptides contain two poly(ethylene glycol)5000 chains while non-cross-linked peptides contain a single poly(ethylene glycol)5000 chain. The two populations of peptides can be separated by gel filtration chromatography. This procedure has been shown capable of isolating cross-linked peptides using glutathione, lysozyme, chemically cross-linked hemoglobin, and neurofibrillary tangles isolated from the brain of a case of Alzheimer's disease.