RESUMEN
4'-Phosphopantetheinylation (4PPTylation) of proteins, which is derived from the hydrolysis of coenzyme A (CoA), is an essential post-translational modification participating in biosynthetic and metabolic pathways. However, due to the lack of specific recognition ligands as well as the shortage of sensitive analytical tools for single-cell analysis, the in-depth exploration of new cellular functions and mechanisms of protein 4PPTylation has been much hampered. In this study, we rationally engineered CoA-imprinted Raman nanotags for the specific recognition of 4PPTylation and thereby developed a molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA) for facile probing the 4PPTylation of ALDH1L1 in single cells. The molecularly imprinted nanotags exhibited excellent binding properties, giving a dissociation constant of 10-6 M and cross-reactivity values of less than 10%. The MIP-based PISA enabled the specific and sensitive detection of the level of 4PPTylated ALDH1L1 in single living cells. Particularly, monitoring of the fluctuation of 4PPTylated ALDH1L1 in single cells under simulation by an inhibitor (methotrexate) that acts on a different metabolism pathway was achieved, implying possible crosstalk between two different pathways in folate metabolism. Thus, the imprinted Raman nanotags-PISA provides a promising analytical tool with a single-cell resolution for exploring new functions and elucidating their mechanisms of protein 4PPTylation.