Page 135 - Tyrosine-Based Bioconjugations - Jorick Bruins

Inducible, Selective Labelling of Proteins via Enzymatic Oxidation of Tyrosine
4. Either cpTCO-bearing or BCN-bearing probes can be used for SPOCQ. For encompassing all possible variations, we simply call them probes. Probe activity can be tested with mass spectrometry, NMR or other methods. Chemical testing of probe activity can be performed by dissolving tert-butyl quinone in a mixture of 1:1 (v/v) MeOH/water, then adding a slight excess of BCN or cpTCO label. The yellowish colour should fade within seconds.
5. Due to the fact that not all probes dissolve well in water, it is advisable to maintain a concentration of approximately 10 vol.% DMSO. Therefore, different protein concentrations might require different concentrations of functional probes to be added. It is advisable to make a stock solution of maximum concentration. As is the case with the protein, more concentrated stock solutions of probes are also advisable.
6. Generally, DMSO is chosen as the co-solvent for the reaction. While it is not necessary for the reaction to proceed, it improves the solubility of more apolar probes. DMF or DMA can also be used as a co-solvent and likely other non-reactive solvents can be used (e.g. propylene glycol). However, a high concentrations of co-solvent (>20%) can affect mushroom tyrosinase’s reaction rate and stability.13 In the case of water-soluble probes the co-solvent can be omitted altogether.
7. Mushroom tyrosinase should be stored in buffer without sodium chloride, due to chloride ions slowly inhibiting its catalytic activity.12 Generally, phosphate buffer is used, which consists of 50 mM sodium phosphate (monobasic) adjusted to the desired pH using 1.0 M NaOH. Mushroom tyrosinase activity can be tested by dissolving L-tyrosine in 9:1 (v/v) water/DMSO, followed by adding some of the mushroom tyrosinase stock solution. The mixture should turn visibly brown within a few minutes.
8. Mushroom tyrosinase can be purchased from Sigma-Aldrich and can be used without any purification, i.e. 10 mg/mL of mushroom tyrosinase corresponds to 10 mg of lyophilized enzyme dissolved in 1 mL buffer.
9. Generally select a membrane with a MWCO that is 2-3 times smaller than the molecular weight of the protein.
10. A mass spectrometer can be attached to this system to allow LC-MS. In that case, TFA is swapped out for formic acid (FA) for the two HPLC buffers as TFA can interfere with the MS signal.
11. Mushroom tyrosinase should be added to the reaction last, since the oxidation is initiated upon addition, since the presence of reactive quinone species can lead to side-reactions with the side-chains of lysine, histidine and cysteine residues.
12. Mushroom tyrosinase stoichiometry is important for the reaction rate. Although ~1% of enzyme is sufficient for complete conversion, generally 5–10% of mushroom tyrosinase is added to limit the reaction time and minimize side-reactions.
13. The reaction time of SPOCQ varies significantly from protein to protein, depending on the accessibility of the G4Y-tag for the oxidation by mushroom tyrosinase. It is advised to execute a time-resolved reaction series and measure the conversion by HPLC or SDS-PAGE.
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