Propargylamine hydrochloride

The constructs pTXB1-ubiquitin^1–75 pTXB1-LC3B^1–119 were used to express ubiquitin or LC3B as a C-terminal intein fusion protein as described in ref. ^{33 (link)}. In brief, the fusion protein was affinity purified in buffer A (20 mM Hepes, 50 mM sodium acetate, pH 6.5, 75 mM NaCl) from clarified lysates using Chitin Resin (New England Biolabs) following the manufacturer's protocol. On-bead cleavage was performed by incubation with cleavage buffer (buffer A containing 100 mM MesNa (sodium 2-mercaptoethanesulfonate)) for 24 h at room temperature (RT). Resin was washed extensively with buffer A and pooled fractions were concentrated and subjected to size exclusion chromatography (HiLoad 16/600 Superdex 75) with buffer A. To synthesize Ub/LC3B-PA, 300 µM Ub/LC3B-MesNa were reacted with 600 µM propargylamine hydrochloride (Sigma Aldrich) in buffer A containing 150 mM NaOH for 3 h at RT. Unreacted propargylamine was removed by size exclusion chromatography and Ub/LC3B-PA was concentrated using VIVASPIN 20 Columns (3 kDa cutoff, Sartorius) flash frozen and stored at −80 °C.

All activity-based probes used in this study were expressed as C-terminal intein fusion proteins as described previously^{63 (link)}: In brief, the fusion proteins were affinity purified in buffer A (20 mM HEPES, 50 mM sodium acetate pH 6.5, 75 mM NaCl) from clarified lysates using Chitin Resin (New England Biolabs) following the manufacturer’s protocol. On-bead cleavage was performed by incubation with cleavage buffer (buffer A containing 100 mM MesNa (sodium 2-mercaptoethanesulfonate)) for 24 h at room temperature (RT). The resin was washed extensively with buffer A and the pooled fractions were concentrated and subjected to size exclusion chromatography (HiLoad 16/600 Superdex 75 pg) with buffer A. To synthesize the propargylated probe, 300 µM Ub/Ubl-MesNa were reacted with 600 µM propargylamine hydrochloride (Sigma Aldrich) in buffer A containing 150 mM NaOH for 3 h at RT. Unreacted propargylamine was removed by size exclusion chromatography and the probe was concentrated using VIVASPIN 20 Columns (3 kDa cutoff, Sartorius), flash frozen and stored at −80 °C. The NEDD8-PA was a kind gift from David Pérez Berrocal and Monique Mulder (Department of Cell and Chemical Biology, Leiden University).

All ubiquitin activity-based probes used in this study were expressed as C-terminal intein fusion proteins as described previously^{26 (link)}. The fusion proteins were affinity purified in buffer A (20 mM HEPES, 50 mM sodium acetate pH 6.5, 75 mM NaCl) from clarified lysates using Chitin Resin (New England Biolabs) following the manufacturer’s protocol. On-bead cleavage was performed by incubation with cleavage buffer (buffer A containing 100 mM MesNa (sodium 2-mercaptoethanesulfonate)) for 24 h at room temperature (RT). The resin was washed extensively with buffer A and the pooled fractions were concentrated and subjected to size exclusion chromatography (HiLoad 16/600 Superdex 75 pg) with buffer A. To synthesize Ub-PA, 300 µM Ub-MesNa were reacted with 600 µM propargylamine hydrochloride (Sigma Aldrich) in buffer A containing 150 mM NaOH for 3 h at RT. Unreacted propargylamine was removed by size exclusion chromatography and Ub-PA was concentrated using VIVASPIN 20 Columns (3 kDa cutoff, Sartorius), flash-frozen, and stored at −80 °C. The conversion of Ub-MesNa to Ub-PA was confirmed by intact mass analysis.