Screening of a series of carboxylic acid labeling reagents implied that reactivity and selectivity control of the bioconjugation is possible through electronic and steric alterations (Figures ). We have tested carboxylic acid compounds to assess both the electronic and steric effects on reactivity and selectivity. While some carboxylic acids are solids under ambient conditions such as pentynoic acid (1e), we discovered that the labeling process proceeds efficiently in a 4-5 M solution of the acid in a nonaqueous solvent such as acetonitrile and ionic liquid (Figures S35, S61-62). Whereas overall modification efficiency varied by a type of carboxylic acid under the tested conditions, we did not observe clear trends of the selectivity changes toward serine over threonine through the introduction of heteroatoms at the α-position as well as cyclic structures for electronic and steric perturbations. The acylation product formation was also observed by the acids with bioorthogonal handles such as azide and terminal alkyne groups, although the reaction with azidoacetic acid showed an unidentified byproduct in the LC-MS analysis (Figure ). The feasibility of the acylation process with 4-pentynoic acid (1e) and subsequent copper-catalyzed azide-alkyne cycloaddition reaction of the acylated group was confirmed as well (Figures ). It is noteworthy that typical reactive acylating reagents (i.e., acyl chlorides and acid anhydrides) were reported to modify even tyrosine residues in addition to serine, underscoring the importance of the acidic catalysis. The acid-catalyzed acylation processes were able to modify a range of polypeptide substrates, enabling the installation of a bioorthogonal handle for imaging purposes (Figure ). Trifluoroacetic acid (TFA) was chosen as a catalyst based on the results of catalyst screenings with peptide and protein as substrates (Table -S3, Figures ) in addition to its compatibility with LC-MS analysis. The acylation products were observed for polypeptides such as insulin (the number of serine residues: 3), ubiquitin (3), ribonuclease A (15), lysozyme (10), β-casein (16), and concanavalin A (31) as shown in Figure and Figures . We previously demonstrated that ionic liquids can be conducive for retaining protein structure and activity, and we employed imidazolium-based ionic liquid (ethyl-methylimidazolium tetrafluoroborate) as a solvent for labeling with a solid alkyne-tagged carboxylic acid 1e. Using a blot membrane-based alkyne detection method, positive fluorescence responses were observed for modification of model proteins concanavalin A, ribonuclease A, and Herceptin, suggesting the successful installation of the alkyne tag through the acylation process (Figures ). The modification of concanavalin A and ribonuclease A was also confirmed by mass spectrometry (Figure ). While the trypsin digestion and subsequent tandem mass spectrometry analysis of modified protein showed some adduct formation on lysine and tyrosine residues to a certain extent (Figures ), peaks of modified serine peptide fragments were substantially high in the mass count (Table ), which also supports our hypothesis about the alkylalcohol selectivity of the method. Although the intact MS analysis did not indicate the adduct formation of TFA catalyst to model protein and peptide substrates, it should be noted that some TFA adducts were observed in the proteomics study to a lesser extent.