Probing Protein 4′-Phosphopantetheinylation in Single Living Cells

Abstract

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 CoAimprinted 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 crossreactivity 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.

Recent Advances in Rational Design and Engineering of Signal-Amplifying Substrates for Surface-Enhanced Raman Scattering-Based Bioassays

“…It combines in vivo microextraction for specific enrichment of target proteins from individual cells, and PERS for highly sensitive detection of low-copy-number proteins (Figure 6B). This approach holds great promise for advancing our understanding of cellular heterogeneity and the functions of low-copy-number proteins in biological processes and disease states [114,115]. PERS-based bioassays are not limited to a single-cell analysis.…”

Molecular Imprinting-Based SERS Detection Strategy for the Large-Size Protein Quantitation and Curbing Non-Specific Recognition

“…Methods in the second category depend on protein labeling with the aid of SERS nanotags. In these systems, captured proteins by imprinted cavities of MIPs are sandwiched between the plasmonic substrate and SERS nanotags, leading to the generation of intense SERS signal . Concerning this methodology, the extent of SERS enhancement is distance-dependent and thus exhibits a strong dependency on the molecular size of protein.…”

“…Despite extensive development in smart imprinting strategies aimed at enhancing the recognition performance of MIPs, even advanced MIPs still encounter unavoidable issues with nonspecific recognition. , Consequently, the potential of state-of-the-art MI-SERS sensors for real-world applications is hindered. In practice, nonspecific recognition occurs due to the undesired perching of matrix species into the imprinted cavities and surface adsorption of nontarget molecules on the imprinted materials .…”

“…These limitations collectively highlight the lack of ideal MI-SERS sensing mechanisms for large-size protein quantitation. Despite extensive development in smart imprinting strategies aimed at enhancing the recognition performance of MIPs, 11 even advanced MIPs still encounter unavoidable issues with nonspecific recognition. 18,19 Consequently, the potential of state-of-the-art MI-SERS sensors for real-world applications is hindered.…”