C4 column

The peptides APX, APX-17, and APX-12 were prepared by FMOC solid phase synthesis. Synthesis was performed on rink-amide resin using Fmoc-protected amino acids, HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), and DIEA (N,N-Diisopropylethylamine) at a 1:1:2 ratio for couplings. Deprotections were performed using a solution of 1:4 v:v: piperidine in DMF (N,N-Dimethylformamide). Cleavage was performed by mixing the resin with a cocktail of TFA (trifluoroacetic acid):TIS (triisopropylsilane):H2O:ethanedithiol (92.5:2.5:2.5:2.5) for ~2 h followed by filtration and peptide precipitation in cold diethyl ether. Remaining peptides were purchased from GenScript (Piscataway, NJ, USA). All peptides were purified by reverse phase high-performance liquid chromatography using a linear gradient of solvent A (H20 with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA). Peptides were separated on an Agilent (5 μm 9.4 × 250 mm) C4 column. Fractions were monitored using UV absorbance at 220 and 280 nm and peptide identity was confirmed using ESI-MS (Agilent 1100 Series LC/MSD Trap, Santa Clara, CA, USA). Purified peptides were lyophilized, reconstituted in an ethanol water mixture and stored at 4 °C. Peptide stock concentration was determined spectrophotometrically, as described previously [31 (link),32 (link)].

Pure mTFP1 and GFP-nanobodies were analysed by mass spectrometry (maXis HD ESI-TOF, Bruker). The sample was injected into a high-performance liquid chromatography (Agilent Technologies, C4 column, column volume of 5 mL) and separation was performed at a constant flow rate of 0.5 µL/min and a gradient of 80% acetonitrile and 0.1% formic acid for 8 min. Fractions were recorded according to the standard procedure.