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<p>Tag-free protein modification by Escherichia coli lipoate ligase A (LplA) enables the installation of an azide handle onto native proteins, followed by copper-free click labeling with fluorescent probes. A practical analytical challenge in this workflow is the intact-level separation of unmodified protein, dye-conjugated protein, and residual free dye without enzymatic digestion or denaturation-based subunit analysis. Here, we describe a reversed-phase high-performance liquid chromatography with photodiode-array detection (RP-HPLC-PDA) method for intact analysis of Cy3-labeled human serum albumin (HSA) and bovine serum albumin (BSA) conjugates generated by LplA-mediated azide installation and DBCO-Cy3 labeling. Several reversed-phase column formats were compared under acidic water/acetonitrile conditions, including C18, C8, polystyrene/divinylbenzene, and polyphenyl column formats. The C8 column showed insufficient retention of intact HSA, whereas the C18 column showed severe late or incomplete elution. The polystyrene/divinylbenzene column provided only partial retention under the tested conditions. Under the common screening conditions used in this study, the BioResolve RP mAb polyphenyl column provided the most useful chromatographic profile among the tested columns. Because the tested columns differed not only in bonded-phase chemistry but also in pore size, particle size, support material, and particle morphology, the observed performance should not be attributed solely to polyphenyl surface chemistry. As an application of the method, HSA and BSA showed different apparent Cy3/protein signal ratios despite their related folds. This difference was examined using an exploratory lysine solvent-exposure analysis. The method does not by itself assign site-specific modification but provides a practical intact RP-HPLC-PDA platform for monitoring native protein-dye conjugates and selecting samples for subsequent peptide mapping or mass spectrometric characterization.</p>

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Polyphenyl reversed phase chromatography separates Cy3 labeled serum albumins from unmodified proteins and free dye

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  • 发表日期：

    2026年8月4日

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  • Shunsuke Yamazaki,
  • Kazutoshi Takahashi,
  • Yutaka Matsuda

摘要

Tag-free protein modification by Escherichia coli lipoate ligase A (LplA) enables the installation of an azide handle onto native proteins, followed by copper-free click labeling with fluorescent probes. A practical analytical challenge in this workflow is the intact-level separation of unmodified protein, dye-conjugated protein, and residual free dye without enzymatic digestion or denaturation-based subunit analysis. Here, we describe a reversed-phase high-performance liquid chromatography with photodiode-array detection (RP-HPLC-PDA) method for intact analysis of Cy3-labeled human serum albumin (HSA) and bovine serum albumin (BSA) conjugates generated by LplA-mediated azide installation and DBCO-Cy3 labeling. Several reversed-phase column formats were compared under acidic water/acetonitrile conditions, including C18, C8, polystyrene/divinylbenzene, and polyphenyl column formats. The C8 column showed insufficient retention of intact HSA, whereas the C18 column showed severe late or incomplete elution. The polystyrene/divinylbenzene column provided only partial retention under the tested conditions. Under the common screening conditions used in this study, the BioResolve RP mAb polyphenyl column provided the most useful chromatographic profile among the tested columns. Because the tested columns differed not only in bonded-phase chemistry but also in pore size, particle size, support material, and particle morphology, the observed performance should not be attributed solely to polyphenyl surface chemistry. As an application of the method, HSA and BSA showed different apparent Cy3/protein signal ratios despite their related folds. This difference was examined using an exploratory lysine solvent-exposure analysis. The method does not by itself assign site-specific modification but provides a practical intact RP-HPLC-PDA platform for monitoring native protein-dye conjugates and selecting samples for subsequent peptide mapping or mass spectrometric characterization.