Abstract
Proximal multi-site phosphorylation is a critical post-translational modification in protein biology. The additive effects of multiple phosphosite clusters in close spatial proximity triggers integrative and cooperative effects on protein conformation and activity. Proximal phosphorylation has been shown to modulate signal transduction pathways and gene expression, and as a result, is implicated in a broad range of disease states through altered protein function and/or localization including enzyme overactivation or protein aggregation. The role of proximal multi-phosphorylation events is becoming increasingly recognized as mechanistically important, although breakthroughs are limited due to a lack of detection technologies. To date, there is a limited selection of facile and robust sensing tools for proximal phosphorylation. Nonetheless, there have been considerable efforts in developing optical chemosensors for the detection of proximal phosphorylation motifs on peptides and proteins in recent years. This review provides a comprehensive overview of optical chemosensors for proximal phosphorylation, with the majority of work being reported in the past two decades. Optical sensors, in the form of fluorescent and luminescent chemosensors, hybrid biosensors, and inorganic nanoparticles, are described. Emphasis is placed on the rationale behind sensor scaffolds, relevant protein motifs, and applications in protein biology.
Highlights
It has been widely observed that differing phosphorylation patterns can lead to different protein interactions with distinct biological outcomes.[7]
Multisite phosphorylation patterns have been shown to be functionally significant beyond the capability of single site modification.[16]
Proximal phosphorylation events have a strong tendency to arise from the same kinase.[20]
Summary
It has been widely observed that differing phosphorylation patterns can lead to different protein interactions with distinct biological outcomes.[7]. Researchers still incorporate complex protocols with enrichment steps to access these deep levels of the proteome.[45] Phosphorylation state-specific antibodies are widely used in a variety of assay types including enzymelinked immunosorbent assays (ELISA), western blotting and immunoimaging.[46] Many phosphorylation state-specific antibodies are commercially available, and advancements in the production process have shortened the generation time for novel antibodies to as little as 2 weeks.[47] applying this strategy for proximal phosphorylation detection requires a more complex sandwich array format where multiple antibodies detect more than one phospho-site epitope on the same protein, which must be stringently validated to have little interference or non-specific binding.[46] Other reported analytical methods for proximal phosphorylation detection include HPLC separation,[48] impedance spectroscopy,[49] and nanopore sequencing.[50,51] Chemically, multi-phosphorylated peptides are challenging to synthesize using traditional methods, which further hampers their capacity as experimental tools.[52] facile and robust detection of protein phosphorylation remains a challenge for many applications due to method limitations.[53] As such, convenient and high-throughput analytical solutions for studying protein proximal phosphorylation are in great demand. This review will highlight the rationale behind sensor design, analyte detection limits, sequence selectivity for specific proximal motifs and their relevant biological applications
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