Page 105 - Tyrosine-Based Bioconjugations - Jorick Bruins

Engineering of Tyrosine-Containing Peptide Loops Enables Non-terminal Protein Labeling
Figure 3. SDS-PAGE analyses of SPOCQ attempted on (A) Tras-Y1 and (B) Tras-Y2 with varying pH (5.5 or 7.1) and temperature (4 °C or 37 °C). Each condition was performed with only antibody, antibody with mTyr, and antibody with both mTyr and BCN-lis.
Additionally, HPLC spectra were recorded of all the formed products (SI S6.4). A lack of consistency was observed however; while each attempt at SPOCQ yielded a visible peak with a fluorescent signal, the yield was no higher than 10-20% in each case. Furthermore, there did not seem to be an increase in conjugation yield between pH 5.5 and pH 7.1, and all HPLC spectra displayed a significant amount of unidentified peaks.
Optimization of SPOCQ on Tras-Y1 and Tras-Y2 was attempted by varying the concentration of antibody in the reaction, as earlier work indicated that performing SPOCQ at higher concentrations suppresses side reactions.8 Thus, overnight SPOCQ was performed on both Tras- Y1 and Tras-Y2 in PBS pH 5.5 at 4 ˚C with various antibody concentrations, after which SDS-PAGE (Figure 4) and HPLC (SI S6.5) analyses were performed. Both SDS-PAGE and HPLC indicate a significant amount of unidentified bands and peaks for Tras-Y1, which might indicate degradation of the product (Figure 4, lane A – E). Even the sample containing only the antibody seems significantly different compared to the previous gel/chromatogram, which was performed six days before this experiment. While Tras-Y2 does not seem to degrade as much as Tras-Y1, there is a new band at 37 kDa that also displays fluorescence (Figure 4, Lane H – J). Furthermore, none of the conditions shows any improvement in reaction yield based on HPLC analysis.
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