Sequence and Conformational Analysis of Peptide-Polymer Bioconjugates by Multidimensional Mass Spectrometry.

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: l-propargylglycine) and their conjugates with poly(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) interfaced with tandem mass spectrometry (MS2) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IM-MS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS2 experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas phase were largely formed in solution, as corroborated by independent circular dichroism (CD) experiments. The collision cross sections (rotationally averaged forward moving areas) of the random coil and helical conformers of the peptides and their PEG conjugates were simulated for comparison with the experimental values deduced by IM-MS in order to confirm the identity of the observed architectures and understand the stabilizing effect of the polymer chain. C-terminal PEGylation is shown to increase the positive charge density and to solvate intramolecular positive charges at the conjugation site, thereby enhancing the stability of α-helices, preserving their conformation and increasing helical propensity.

Aggregation of poly(acrylic acid)-containing elastin-mimetic copolymers.

Polymer-peptide conjugates were produced via the copper-catalyzed azide-alkyne cycloaddition of poly(tert-butyl acrylate) (PtBA) and elastin-like peptides. An azide-functionalized polymer was produced via atom transfer radical polymerization (ATRP) followed by conversion of bromine end groups to azide groups. Subsequent reaction of the polymer with a bis-alkyne-functionalized, elastin-like peptide proceeded with high efficiency, yielding di- and tri-block conjugates, which after deprotection, yielded poly(acrylic acid) (PAA)-based diblock and triblock copolymers. These conjugates were solubilized in dimethyl formamide, and addition of phosphate buffered saline (PBS) induced aggregation. The presence of polydisperse spherical aggregates was confirmed by dynamic light scattering and transmission electron microscopy. Additionally, a coarse-grained molecular model was designed to reasonably capture inter- and intramolecular interactions for the conjugates and its precursors. This model was used to assess the effect of the different interacting molecular forces on the conformational thermodynamic stability of the copolymers. Our results indicated that the PAA's ability to hydrogen-bond with both itself and the peptide is the main interaction for stabilizing the diblocks and triblocks and driving their self-assembly, while interactions between peptides are suggested to play only a minor role on the conformational and thermodynamic stability of the conjugates.

Top-down mass spectrometry of hybrid materials with hydrophobic peptide and hydrophilic or hydrophobic polymer blocks.

A multidimensional mass spectrometry (MS) methodology is introduced for the molecular level characterization of polymer-peptide (or polymer-protein) copolymers that cannot be crystallized or chromatographically purified. It encompasses electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) coupled with mass analysis, tandem mass spectrometry (MS(2)) and gas-phase separation by ion mobility mass spectrometry (IM-MS). The entire analysis is performed in the mass spectrometer ("top-down" approach) within milliseconds and with high sensitivity, as demonstrated for hybrid materials composed of hydrophobic poly(tert-butyl acrylate) (PtBA) or hydrophilic poly(acrylic acid) (PAA) blocks tethered to the hydrophobic decapeptide VPGVGVPGVG (VG2) via triazole linkages. The composition of the major products can be rapidly surveyed by MALDI-MS and MS(2). For a more comprehensive characterization, the ESI-IM-MS (and MS(2)) combination is more suitable, as it separates the hybrid materials based on their unique charges and shapes from unconjugated polymer and partially hydrolyzed products. Such separation is essential for reducing spectral congestion, deconvoluting overlapping compositions and enabling straightforward structural assignments, both for the hybrid copolymers as well as the polymer and peptide reactants. The IM dimension also permits the measurement of collision cross-sections (CCSs), which reveal molecular architecture. The MS and MS(2) spectra of the mobility separated ions conclusively showed that [PtBA-VG2]m and [PAA-VG2]m chains with the expected compositions and sequences were formed. Single and double copolymer blocks (m = 1-2) could be detected. Further, the CCSs of the hybrids, which were prepared via azide/alkyne cycloadditions, confirmed the formation of macrocyclic structures. The top-down methodology described would be particularly useful for the detection and identification of peptide/protein-polymer conjugates which are increasingly used in biomedical and pharmaceutical applications.

Nanoparticle Formation from Hybrid, Multiblock Copolymers of Poly(Acrylic Acid) and VPGVG Peptide.

Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 × 10-2 or 1.05 × 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through concerted H-bonds and hydrophobic interactions.

Biofunctional paper via the covalent modification of cellulose.

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Responsive Hybrid (Poly)peptide-Polymer Conjugates.

(Poly)peptide-polymer conjugates continue to garner significant interest in the production of functional materials given their composition of natural and synthetic building blocks that confer select and synergistic properties. Owing to opportunities to design predefined architectures and structures with different morphologies, these hybrid conjugates enable new approaches for producing micro- or nanomaterials. Their modular design enables the incorporation of multiple responsive properties into a single conjugate. This review presents recent advances in (poly)peptide-polymer conjugates for drug-delivery applications, with a specific focus on the utility of the (poly)peptide component in the assembly of particles and nanogels, as well as the role of the peptide in triggered drug release.