Figure 3. General workflow of chemical labeling methods to identify interaction partners of SEPs and alt-proteins.

(A) Schematic of APEX2 fusion-based proteomics to identify interaction partners of SEPs and alt-proteins. APEX2 is fused to the SEP of interest, and is expressed in cells. In the presence of biotin-phenol and H₂O₂, APEX2 generates biotin phenoxy radicals, which can label proximal proteins at aromatic amino acid sidechains, enabling their purification with streptavidin and identification with proteomics. (B) Schematic of photo-crosslinking uAA-based proteomics to identify interaction partners of SEPs and alt-proteins. A uAA such as AbK, a lysine analog bearing a diazirine photo-cross-linker, is incorporated into the SEP of interest using amber codon suppression technology in cells; the SEP is also fused to an epitope tag for purification of cross-linked complexes. After photo-irradiation, the diazirine is converted to a reactive carbene, which can insert into C-H bonds on proximal proteins to form a covalent bond. Immunopurification of the cross-linked complexes followed by trypsin digest and mass spectrometry enables identification of interaction partners. (C) Schematic of chemical cross-linking for identification of SEP interaction partners. Bivalent compounds containing two reactive chemical groups bridged by a linker are added to cells or lysates, enabling formation of covalent bonds to nucleophilic amino acid side chains on two interacting proteins, trapping them in a complex. Subsequent trypsin digest, followed by proteomics with smORF/alt-ORF database searching, enables global discovery of SEP and alt-protein complexes in an unbiased manner.