Page 134 - Tyrosine-Based Bioconjugations - Jorick Bruins

Appendix
1. Prior to HPLC analysis, freshly prepare a 0.2 M DTT solution in 0.1 M Tris pH 8.0.
2. Dilute antibody samples to 1 μg/μL antibody and add 2.5 μL DTT stock solution per 10 μg
antibody.
3. Incubate the mixture for 30 minutes at 37 °C in order to reduce the interchain disulfide
bonds. After which the reaction was quenched by adding 30 uL of a mixture of 49:49:2
(v/v/v) MilliQ:acetonitrile (MeCN): FA per 10 μg antibody.
4. HPLC analysis was performed by injecting 10-25 μL of reduced antibody. Analysis was
performed with a flow rate of 0.6 mL/min at 80 °C using a 10 minute linear gradient from 20% to 45% buffer B (with buffer A = 95% MilliQ, 5% acetonitrile, 0.1% trifluoroacetic acid (TFA) and buffer B = 95% acetonitrile, 5% MilliQ, 0.1% TFA).
5. Protein absorption spectra were measured at λ= 215 nm and λ= 280 nm. Another wavelength can be selected if the attached probe has a specific absorbance (e.g. λ= 586 nm for Lissamine i.e. sulphorhodamine B).
Example: After performing the SPOCQ reaction on Tras[HC]G4Y according to the procedure described in section 3.1, DTT denaturation and HPLC analysis can be performed as described in section 2.3.2. This results in a shift in retention time for the heavy chain from 8.47 min to 8.94 minutes (Figure 4C) in case of a conjugation of BCN-lissamine to Tras[HC]G4Y.
Notes
1. Generally, PBS is used for the SPOCQ reaction, which consists of 50 mM sodium phosphate (monobasic) and 150 mM of sodium chloride and is buffered to the desired pH using 1.0 M NaOH. Alternatively, other non-nucleophilic bases such as HEPES or MES can be used.
2. While pH 5.5 is optimal, SPOCQ has been performed at pH ranges from 5.5–8.0 (mushroom tyrosinase is deactivated below pH 5.0).12 Lowering the pH to 5.5 will increase the yield due to a higher level of protonation of the lysine, cysteine and histidine side-chains, which lowers their reaction rates.
3. It is advised to have the protein stock solution fairly concentrated (100–200 μM), with 10 μM as a minimum concentration. Stock solutions can easily be diluted with PBS before reaction if necessary. Some proteins might require a higher concentration to obtain higher yields, as higher concentrations help outcompete intramolecular Michael additions by nucleophilic amino acid residues. A SPOCQ reaction consisting of 50–200 μM protein with 3–10 equivalents of cpTCO or BCN generally results in optimal conversion. If multiple G4Y tags are present on the protein, the equivalents of cpTCO or BCN are calculated versus the amount of G4Y-tags present. For example; two G4Y-tags are present on the Tras[HC]G4Y in section 5.1, and 10 equivalents of cpTCO-lissamine compared to the antibody results in 5 equivalents per G4Y-tag.3
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