Illuminating green fluorescent protein: Characterizing tri-peptide fluorescent chromophore, probing reactivity of cysteines, and unveiling site-directed modifications through mass spectrometry.

Bioconjugation technologies enable covalent attachment of diagnostic or therapeutic effectuators onto biological targets, allowing for the precise delivery of desired drugs to the intended targets with enhanced potency, selectivity, specificity, and prolonged duration of action. As the number of bioconjugation techniques has grown enormously, identification and in-depth characterization of in-process products play a critical role in the development of covalent drug conjugates. This is especially significant in light of the increased complexity of novel biotherapeutics derived from biological matrices. This paper describes liquid chromatography-mass spectrometry (LC-MS/MS)-based studies that have contributed to the development of site-specific genetic incorporation of non-natural amino acids (nnAAs) into proteins. A holistic approach was implemented to characterize a wild type green fluorescent protein (wtGFP) and an enhanced green fluorescent protein (eGFP). By using the wtGFP as a pilot and model system, the reactivity of cysteine residues was investigated under different sample processing conditions, followed by a stability evaluation using intact mass measurement. The subsequent complementary proteolytic peptide mappings were performed to achieve full sequence coverage of the proteins, identification of predominant modifications, and granular details of the fluorescent chromophore. The developed method was successfully applied to isolate the eGFP incorporated with nnAA from cells. This enables the verification of the specific site of nnAA incorporation, and the characterization of complex variants using de novo sequencing techniques. MS studies demonstrated that p-azido-phenylalanine (pAzF) was specifically incorporated into the desired site of eGFP with high efficiency and fidelity.