Fig. 4: A toolkit for analysis of APP processing in the endolysosomal system.

a Schematic overview of proposed APP processing events in the endolysosomal system. APP has an extracellular domain containing E1 (blue), KPI (purple), E2 (orange), juxtamembrane (dark gray), a transmembrane domain (light gray), and a C-terminal domain termed AICD (tan). APP can be delivered to the plasma membrane through a canonical secretion pathway or delivered directly to early endosomes from the Golgi via an AP-4-dependent process. Cleavage of APP by α-Secretase on the plasma membrane constitutes a non-amyloidogenic processing pathway. APP can also be endocytosed into EEA1 and RAB5-positive vesicles that contain BACE1 and γ-Secretase. These organelles can mature to lysosomes that contain TMEM192 in the membrane and have been reported to contain higher levels of PSEN2²⁶, a catalytic component of γ-Secretase. The extent to which processing to form Aβ peptides occurs in the endosome or lysosome is unclear. b Schematic depicting the rational and workflow for detection of half-tryptic peptides for quantification of peptides derived from Aβ40, 42, or 43. The sequence around the APP TM is shown, as well as the location of Aβ trigger peptides used for TOMAHAQ. Synthetic Aβ40, 42, and 43 peptides were digested with trypsin, labeled with TMT-126, mixed with TMT-unlabeled extracts from 293 cells, as well as with trigger peptides previously labeled with TMTsh. c During TOMAHAQ, trigger peptides identified in MS² are used to isolate “target” peptides from synthetic Aβ, which are then subjected to SPS-MS³ to allow reporter ion quantification. MS² fragments used as trigger for the Aβ40 half-tryptic peptide are shown. d Signal-to-noise values for Aβ-derived tryptic and half-tryptic peptides derived from the experiment outlined in panel B (n = 1). e Summary of APP/Aβ trigger peptides, including their locations within the APP protein. Half-tryptic peptides for processing by BACE1 and γ-secretase are shown in red. Several peptides represent isoforms or phospho-forms of APP. f Chromatographic profiles of trigger peptides from MS¹.