Protein triple labelling was demonstrated with CTC-445.2d modified with an N-terminal LPETG, an internal KL-tag and a C-terminal NGLH motif. In the first step, 50 µM protein was C-terminally labelled using 1 µM OaAEP1 in 100 mM HEPES buffer, pH 7, containing 20 mM propargylamine. Next, the reaction was diluted tenfold with 100 mM HEPES buffer, pH 8, and bound to Ni-NTA. After collecting the flowthrough, the protein was N-terminally cleaved with SrtA-7M in the presence of 5 mM propargylamine. Next the protein was buffer-exchanged into 100 mM HEPES buffer, pH 8, using NAP-5, and N-terminally labelled in a reaction containing 30 µM protein, 450 µM TAMRA-LPETGGH and 1 µM SrtA-7M. The SrtA-7M was subsequently removed by rebinding to Ni-NTA and collecting the flowthrough. Next, the reaction was buffer-exchanged via NAP-5 into 100 mM HEPES buffer, pH 8, and the KL-tag labelling was carried out in a reaction containing 50 µM protein, 1 mM biotin-RNGLH, 1 mM NiSO 4 and 1 µM OaAEP1 for 4 h at 25 °C. https://doi.org/10.1038/s41557-024-01520-1