Ab in Lens in Down Syndrome bar = 500 nm. (K) High-magnification electron micrograph of a single Ab-immunoreactive cytoplasmic protein aggregate (arrow) in a lens fiber cell from same Down syndrome lens section shown in Fig. 3J. Multiple immunogold particles detect a single cytoplasmic protein aggregate with longest axial cross-section ,50 nm. Scale bar = 50 nm. (L) Confirmation of anti-Ab antibody specificity. Anti-Ab immunostaining was not detected in Down syndrome lens when probed with immunodepleted anti-Ab antibody. doi:10.1371/journal.pone.0010659.g003 molecular weight of ,4 kDa corresponding to monomeric Ab that was also detected in homogenate of AD brain. The apparent Ab monomer bands detected in DS and AD lens and brain homogenate migrated with the same electrophoretic mobility observed in the single band detected in a sample of purified synthetic human Ab40. Ab monomer and apparent oligomeric species were also detected as weak but distinct bands in normal control lens. Total Ab immunoreactive material detected in the lens of a 2-year-old DS subject was comparable to that detected in lenses from a 57-year-old subject with familial AD and an 85-year-old subject with neuro- pathologically-confirmed late-onset AD (Fig. 6C). The results of the immunoblot analysis provide confirmatory evidence supporting the finding of Ab in DS lenses and also demonstrate an apparent increase in oligomeric Ab and corresponding decrease in monomeric Ab in the lens as a function of advancing age. Down Syndrome Ab Lens Pathology Recapitulated in a Model Lens Protein System In Vitro These data indicate that Ab accumulates in the cytoplasm of supranuclear lens fiber cells in individuals with DS. We previously showed that Ab binds (Kapp ,20 nmol/L) and co-aggregates with aB-crystallin (HspB5) [28], a prototypic molecular chaperone and small heat shock protein [32] that is abundantly present in the lens Figure 4. Peptide sequencing of human Ab from Down syndrome lens. Tryptic digest tandem mass spectrometry sequencing of a ,4 kDa HPLC eluate derived from human Down syndrome lens protein extract. The retention time of the immunopurified HPLC eluate used for sequencing was identical to synthetic human Ab. The red shade box denotes the detected 12-residue internal tryptic peptide sequence 17LVFFAEDVGSNK28 that uniquely identifies human Ab. We detected a second unique tryptic peptide 6HDSGYEVHHQK16 in another analysis (purple underline). doi:10.1371/journal.pone.0010659.g004 PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10659