The compatibility of the acidic bioconjugation conditions with protein substrates was demonstrated through enzyme activity assays and immunofluorescence experiments (Figure ). To estimate the effects of an acidic nonaqueous medium on the activity of an enzyme, lysozyme (a glycosyl hydrolase) was incubated in acetic acid at 37 °C for 24 hours and then subjected to a fluorescence-based enzyme-activity assay. The acid-treated lysozyme showed a comparable level of fluorescence intensity as the positive control sample treated in an aqueous buffer (Figure ), and thus the retained enzymatic activity in aqueous buffer after the acid treatment was indicated. We also tested the binding activity of a commercial fluorophore conjugate of a glycol-protein binding protein, wheat germ agglutinin (WGA CF®633) to cancer cell lines after incubation in acetic acid without a catalyst (i.e., non-acetylating conditions), and the confocal microscope imaging demonstrated similar fluorescence intensity and staining pattern of the acidtreated sample to those of the positive control protein treated in an aqueous buffer (Figures ). Encouraged by those results of the retention of protein activities in acidic media without the acylation process, we then evaluated the binding activity of modified WGA after the catalytic bioconjugation in the acidic medium as well as subsequent fluorophore attachment through the copper-catalyzed azide-alkyne cycloaddition reaction. The fluorescently labeled protein was characterized by gel fluorescence (Figure ). The localization patterns of the HEK293T cells stained with the prepared fluorophore conjugate proved virtually identical to those by the commercial staining dye (Figures ), and the acidic modification and alkylalcohol tagging processes probably did not have substantial negative impacts on the protein activity. The bioconjugation in the acidic media can offer general alkylalcohol labeling without obvious preferences to active serines in enzymatic pockets (Figure ). The commonly used alcohol-targeting protein labeling reagents, fluorophosphonates, are known to selectively modify the serine residues of the active site of serine proteases. As the fluorophosphonate-based labeling is limited to enzyme active sites, we envisioned that the acylation catalysis may be able to act as a complementary alkylalcohol-targeting method. Incubation of a model serine protease (α-chymotrypsin) and corresponding proenzyme (αchymotrypsinogen A) with fluorophosphonates 2 showed a sole modification toward chymotrypsin whereas the proenzyme remained intact. On the other hand, the acid-catalyzed acylation showed adduct formations in both substrates, indicating the independent nature of the labeling efficiency from the active site status (Figures , S102-S104). Finally, the unique selectivity profile of the acidic catalysis was also demonstrated at a cell lysate level (Figure ). First, the acylation processes in acidic media did not show any noticeable changes to the band shapes and patterns of HEK293T cell lysates in the total stain image from those of the samples in aqueous solutions. Compared to the cell lysate modified by 2, numerous bands showed fluorescence signals for the acylation samples in the presence of the catalyst in the blot analysis, suggesting modification on many types of proteins beyond specific serine proteases. The fluorescence signals with proteins treated with fluorophoshonate 2 were decreased for heat-denatured cell lysates through the loss of the protease activity as reported in the literature. By contrast, the acid-catalyzed acylation did not show an apparent difference by the heat denaturation, further confirming that the acid-catalyzed protein modification is independent of the serine protease activities. The band pattern of cell lysate of HEK293T modified with fluorophosphonate alkyne 2 was similar to the reported gel-based activity protein profiling results with fluorophosphonate-fluorophore conjugates (Figure ). These results indicate that the acid-catalyzed acylation method would serve as a complementary serine-targeting method to the enzyme-specific labeling with fluorophosphonates.