Extended Data Figure 3: Mass spectrometry analysis of Aη peptides.

a, b, After removal of sAPP-α from conditioned media of CHO 7PA2 cells using appropriate centricon filters, the flow-through was used to isolate Aη peptides by immunoprecipitation. Synthetic peptides (1 ng per lane) were loaded to indicate the respective sizes of Aη-α and Aη-β. Aη peptides were captured with antibodies 9476M and 9478D directed against the putative N-terminal epitopes (Supplementary Table 1), 2E9 against a middle domain of Aη and 2D8 (which also immunoprecipitates amyloid-β). 2D8 detection (a) revealed that all antibodies captured peptides positive for the N-terminal part of the amyloid-β domain in a molecular mass range of synthetic Aη-α between 12 and 16 kDa. The same samples analysed with 2E9 (b) confirmed the presence of Aη in all samples, with the lowest levels when precipitated with 9476M. Note that unlike 2D8 antibody, 2E9 allows the additional detection of Aη-β. (Asterisks denote IgG.) c, A heat map of peptides identified after analytic proteolysis by mass spectrometry analysis, with 7PA2 supernatants immunoprecipitated with 2D8, 2E9, 9476M and 9478D antibodies. Arrows indicate peptides that start exactly with the amino acid sequence C-terminal of the respective cleavage sites of the η-secretase, β-secretase or α-secretase site. Chy, chymotrypsin; LysC, protease LysC; tryp, trypsin.