Moreover, the effectiveness of the modification was primarily corroborated through SDS-PAGE and RP-HPLC analysis using phenyl resin (Table and figure S1-S9 in SI). Notably, UVbased determination of the reaction conversion of protein conjugates was effectively conducted using RP-HPLC under non-reducing conditions, highlighting its utility. This approach is particularly relevant as UV-based analysis is often considered to provide a more accurate conversion rate than LC-MS analysis. The latter can be influenced by ionization efficiency [37]. However, it was observed that hydrophobic interaction chromatography gave less effective chromatographic separation for this purpose. Given these considerations, RP-HPLC analysis of intact conjugates, previously reported for ADCs , is also emerging as a viable method for the analysis of protein conjugates. This advancement underscores the versatility and applicability of RP-HPLC in protein conjugate analysis. The initial column screening was performed using HSA and human lysozyme respectively. In the mobile phase system consisting of acetonitrile and water, it was observed that neither HSA nor lysozyme exhibited adherence to the C8 column, resulting in their complete elution without retention. In contrast, on the C18 column, excessive adsorption was observed, hindering the separation of proteins from Cy3, which only eluted in 100% acetonitrile. However, selection of phenyl resin enabled effective separation between Cy3 and the proteins, leading to its use in analyzing the conjugates. Under the employed HPLC conditions, unreacted Cy3 compounds were eluted before the proteins, thus ensuring comprehensive separation between the unmodified proteins and their modified counterparts. Therefore, the use of the phenyl column for HPLC analysis, in conjunction with SDS-PAGE, is deemed suitable for future in situ analyses. For the determination of the modification rate through area in HPLC chromatograms, standard curves were established using Cy3 solutions at varying concentrations, specifically targeting the 570 nm wavelength (Figure in SI). Following this, the conjugates were quantitatively analyzed, and their respective modification rates were derived from the area values obtained at the 570 nm wavelength. The results obtained from RP-HPLC demonstrated greater quantitative precision than the FI measurements from SDS-PAGE, yet the trends identified in SDS-PAGE aligned with these findings (Figure ). In this study, the modification rates of various proteins by LplA were quantitatively translated into dye-to-protein ratio (DPR) values. The highest DPR was observed in HSA at approximately 4, followed by BSA at 1, OVT at 0.7, and transferrin at about 0.5. The differences in these modification rates and LplA's substrate selectivity among proteins were then examined. Prior research has shown that LplA preferentially modifies Lys188 in the antibody trastuzumab, as determined by peptide mapping with three different enzyme digestions. Based on this, the modification rates of the current study's proteins were analyzed with a focus on solvent-accessible surface area (SASA indicating it is a spatially exposed lysine. This suggests that proteins lacking lysines with SASA values around 160 or higher are unlikely to undergo significant LplA-mediated modification. A comprehensive SASA analysis of all proteins was conducted for comparison (Table ).