Ab in Lens in Down Syndrome Figure 2. Stereo images demonstrating mature supranuclear lens pathology in Down syndrome and Alzheimer’s Disease. (A) Characteristic circumferential supranuclear cataract in the lens of a 64-year-old male subject with Down syndrome. This distinctive cataract is evident as an annular half-toroid band of opacification in the deep cortical and supranuclear subregions of the lens. This same lens specimen is presented as a slit lamp biomicrocopic image (Fig. 1H) and as a stereo image pair (with intact zonule fibers, Fig. S1). This dramatic Down syndrome cataract is phenotypically comparable to the incomplete subequatorial supranuclear cataract observed in the lens of a 76-year-old male subject with advanced Alzheimer’s disease (B). These distinctive supranuclear cataracts are not observed in age-normal control subjects. See text for details. doi:10.1371/journal.pone.0010659.g002 immunodepleted detection antibody (Fig. 3L). These histopatho- logical findings are identical to those previously described for lens pathology in late-onset sporadic AD [28]. Identification and Biochemical Characterization of Ab in Down Syndrome Lens Definitive identification of human Ab in DS lens was accomplished by tryptic digest tandem mass spectrometry peptide sequencing. Biochemical sequencing analysis was conducted on small molecular weight eluates derived from HPLC fractionation of human DS lens protein extract (Fig. 4). In positive electrospray mode, the LC-MS/ MS spectrum detected two tryptic peptide signals corresponding to 17LVFFAEDVGSNK28 and 6HDSGYEVHHQK16 that uniquely identify human Ab. The detected fragments did not derive from the larger amyloid precursor protein (APP) as sequencing was performed on eluates with retention times corresponding to monomeric Ab (,4 kDa). In order to establish the tissue concentration of Ab in the lens, we first investigated the relative efficiency of sodium dodecyl sulfate compared to formic acid extraction performed on protein extracts obtained from human lens homogenates (Fig. 5). Formic acid demonstrated a far superior extraction profile relative to sodium dodecyl sulfate for both Ab1-40 and Ab1-42. ELISA results (Fig. 6A,B) demonstrated elevated lens and brain tissue levels of the major human Ab isoforms (Ab40 and Ab42) and total Ab that were consistently elevated in DS compared to normal controls and comparable to the values previously reported in AD [28]. Immunoblot analysis of DS lens extract (Fig. 6C) revealed a prominent Ab-immunoreactive band that migrated with an apparent PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10659