FIGURE 1.

γ-Glutamyl-ϵ-lysine cross-links in brain fractions accumulate in α-synuclein immunopositive chaotrope-insoluble inclusions/Lewy bodies. The relative abundance of GGEL was determined by enzymatic hydrolysis of sample proteins in ¹⁸O-water and comparison with light internal GGEL standard by mass spectrometry. GGEL was related to total amino acid content. A, GGEL accumulation associates with regions affected by degenerative changes in human brain specimen affected by Lewy body dementia (LBD), Alzheimer disease, or in age-matched controls (CTRL). Over 50% of total tissue GGEL content was recovered by either anti-ubiquitin or anti-α-synuclein affinity isolation from chaotrope-, detergent-, and thiol-insoluble brain proteins (B), indicating that most of the cross-linked inclusions expose both antigens in AD and LBD. Lewy bodies were exhaustively digested with proteinase K. Residual proteins remaining after digestions were dissolved in guanidine HCl and co-precipitated with a different protein (egg albumin) to disrupt β-sheet amyloid arrays between proteins. The GGEL density in proteinase K-resistant fractions is by an order of magnitude lower than in the proteinase-digestible part (C). Bars represent the means of duplicate determinations from three different donors. Asterisks and hatch marks indicate significant (p < 0.05) differences from the corresponding controls and AD samples, respectively.