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.
Chen
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Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Eswaran
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D
Smith
M A
MA
Perry
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G
Anderson
V E
VE
D000544:Alzheimer Disease / Q000473:pathology; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D016874:Neurofibrillary Tangles / Q000473:pathology*; D016900:Neurofilament Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D016900:Neurofilament Proteins; C117929:PEG-O-succinyl-O-Su; D010455:Peptides; D013388:Succinimides; D011092:Polyethylene Glycols; D008239:Lysine