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The amyloid-β protein precursor (AβPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AβPP generates the neurotoxic amyloid-β peptide (Aβ). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AβPP. The C-terminal tail of AβPP was fluorescently labeled using a SNAP-tag, while the Aβ region of AβPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AβPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AβPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AβPP variants was validated with ELISA and immunoblotting, and seven AβPP mutants that showed similar cleavage pattern as wild-type AβPP were identified. The AβPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AβPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aβ and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aβ with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AβPP.

Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products