Abstract
The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Here we report a new switchable two-dimensional (2D) supramolecular polymer that serves as an ideal platform for the enrichment of phosphopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, thus resulting in an excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real biosamples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.
Highlights
Over the past few decades, discrete supramolecular coordination complexes (SCCs) with well-defined size, shape, and geometry have been widely constructed through coordination-driven self-assembly.[20−25] These systems have found applications in molecular recognition, sensing, catalysis, biomedicines, and other areas.[26−30] More recently, significant progress has been made on the functionalization of coordination metal complexes to construct stimuli-responsive supramolecular polymers which combine the advantages of well-defined supramolecular coordination complexes and polymeric materials, though either hierarchical self-assembly or postassembly polymerization.[31−39] Motivated by our previous studies on discrete metallacycles[40] and phosphate binding,[41,42] we envisioned that a metallacyclic scaffold with well-defined shape and size may provide an ideal platform to construct polymers that can work as affinity reagents for the recognition of PPs
The hexagonal metallacycle 3 was prepared in quantitative yield by mixing the 120° dipyridyl donor 1 with 120° platinum acceptor 2 in a 1:1 ratio (Figure 1a and Scheme S1)
Multiple peaks observed in both gel-permeation chromatography (GPC) and dynamic light scattering (DLS) analysis (Figure S8) provided evidence supporting a broad mass and size distribution of polymer 5 from tens up to hundreds of kDa
Summary
Anion recognition and extraction have received considerable attention during the last few decades since anionic species play many important roles in a number of areas including biology, pharmacy, industry, and environmental sciences.[1,2] To date, a wide variety of functional groups and molecules have been exploited to selectively recognize, respond to, or sense anionic species.[3−7] Recent results indicate that polymeric systems with bona f ide anion recognition features can offer special advantages for the development of smart materials with applications in anion recognition and extraction.[8−11] comprehensive analysis and identification of anionic biomolecules such as phosphorylated peptides (PPs) remain a challenge owing to anions’ high hydration and PPs’ low abundance as well as significant signal suppression by nonphosphorylated peptides.[12−14] Anionic biomolecules such as PPs are closely associated with a number of human diseases, including Alzheimer’s disease and cancer.[15−17] the development of supramolecular materials that can controllably bind and release PPs under aqueous conditions has numerous potential bioanalytical applications. Four N-terminal fluorescein-labeled model peptides with identical amino acid sequences differing only in the number of phosphate groups (serine mono-, di-, tri-, and tetra-PPs, abbreviated as 1SP−4SP, Figure 4c) were prepared to study the binding affinity of polymer 5 for PPs. Fluorescence titration experiments, in which Ka values were elucidated according to intensity changes in the maximum emission peak, were conducted with the four model peptides and different compounds including polymer 5, metallacycle 3, and related compounds ligand 1 and unit 6 (Figure S40). No obvious change of film morphology and thickness was observed upon interaction with nonphosphorylated peptides NP1 and NP2 (Figures S68 and S69) These results further confirmed satisfactory selectivity and high adsorption capacities of polymer 5 toward PPs. Theoretical calculations of the possible binding modes and binding energies between polymer 5 (metallacycle 3 was considered as the monomer unit of polymer 5) and different model peptides suggested that the skew-crossing mode and skew-clinging mode are the dominant modes of interaction (Figure S70 and Table S3). Article phosphorylation (e.g., histidine phosphorylation), which is widespread in prokaryotes.[58]
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