Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Strategies for formulation of effervescent granules of an herbal product for the management of typhoid fever.

Herbal medicines are currently being adopted as alternatives to orthodox medicines for the management of drug-resistant and emerging multidrug-resistant microbial strains of various diseases, including typhoid fever. A herbal decoction, MA 001, manufactured by the Centre for Plant Medicine Research (CPMR), has been used for the treatment of typhoid fever for at least two decades in Ghana with desirable outcomes. MA 001 is formulated from Citrus aurantifolia, Spondias mombin, Latana camara, Bidens pilosa, Trema occidentalis, Psidium guajava, Morinda lucida, Vernonia amygdalina, Persea americana, Paulina pinnatta, Momordia charantia and Cnestis ferruguinea medicinal plants. The low palatability and compliance to treatment due to the bulky nature of the decoction poses challenges in its optimum use. This study sought to design and formulate the therapeutic components of the aqueous herbal decoction of MA 001 into an optimal solid dosage form of effervescent granules to improve the delivery of MA 001 as well as increase patient compliance and convenience of product handling. The methods involved pre-formulation studies on the suitability of effervescent vehicles, formulation and evaluation of effervescent granules for drug excipient interactions using high performance liquid chromatography analysis. The findings indicate that the effervescent granules were suitable for use in the delivery of the therapeutic constituents for the treatment of typhoid fever as done with the decoction due to minimal herbal extract-excipient interaction.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody-Gold Nanoparticle Conjugates.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles.

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein-AuNP interaction may result in the formation of less desirable protein-AuNP aggregates. Therefore, it is imperative to investigate the protein-AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein-AuNP interactions strengthen over 24 h, after which the protein-AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody-nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.

Role of Free Thiol on Protein Adsorption to Gold Nanoparticles.

Protein-gold nanoparticle (AuNP) bioconjugates have many potential applications in nanomedicine. A thorough understanding of the interaction between the protein and the AuNP is critical to engineering these functional bioconjugates with desirable properties. In this work, we investigate the role of free thiols presented by the protein on the stability of the protein-AuNP conjugate. Human serum albumin (HSA) was modified with 2-iminothiolane (Traut's reagent) to introduce additional thiols onto the protein surface, and three variants of HSA were synthesized to present 1, 5, and 20 free thiols by controlling the molar excess of the chemical modifier. Protein exchange studies on AuNPs were conducted using these HSA species and an IgG antibody which exhibited 10 free thiols. Antibody-AuNP conjugates were synthesized, purified, and dispersed in solutions containing each of the HSA species. No protein exchange was detected with the HSA or modified HSA containing 5 thiols; however, 85% of the antibody was displaced on the AuNP surface by the extensively thiolated HSA presenting 20 free thiols. Furthermore, the impact of the protein adsorption sequence was probed in which each of the HSA species were preadsorbed onto the AuNP and dispersed in a solution of antibody. The antibody fully displaced the HSA with a single thiol from the AuNP within 3 h, required 24 h to completely displace the modified HSA containing 5 thiols, and was unable to displace the modified HSA containing 20 thiols. These results indicate that the number of Au-S interactions governs the binding interaction between the protein and the AuNP. This work provides further insight into the protein-AuNP binding mechanism and identifies important design principles for engineered proteins to optimize bioconjugates.

pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles.

Novel detection strategies that exploit the unique properties of gold nanoparticles (AuNPs) hold great promise for the advancement of diagnostic testing. Fundamental to many of these nanoparticle-enabled techniques is the immobilization of antibodies onto the AuNP surface to afford selective binding to target species. Orientation and loading density of the immobilized antibodies govern Fab accessibility and are critical to the analytical performance. Here, we use pH to systematically control the surface charge distribution on an antibody and investigate the impact of protein charge on adsorption to AuNPs. Nanoparticle tracking analysis (NTA) is used to measure the adsorption dynamics of anti-horseradish peroxidase antibody (anti-HRP) onto AuNPs at different pHs. NTA enables in situ measurement of antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter ( DH) of the AuNPs as a function of antibody concentration. The adsorption affinity, protein layer thickness, and binding cooperativity at each pH are extracted from the best fit of the adsorption isotherms to the Hill-modified Langmuir equation. Our data show a monolayer of antibody is formed at saturation at pHs 7.5, 8.0, and 8.5, and no difference in anti-HRP-AuNP binding constants is observed in this pH range ( Kd ∼11 nM). However, the increase in DH of the AuNPs with adsorbed protein at monolayer coverage is pH-dependent, measuring 13.2 ± 1.1 nm, 9.8 ± 1.0 nm, and 7.4 ± 0.6 nm for pHs 7.5, 8.0, and 8.5, respectively. Moreover, results of an enzyme-mediated assay reveal the antigen-binding capacity of the immobilized anti-HRP antibody is 33 ± 2%, 23 ± 7%, and 18 ± 2% when adsorbed at pHs 7.5, 8.0, and 8.5, respectively. Our data confirm that antibody charge can be altered with pH to modulate and optimize antibody orientation on AuNP. These studies describe our continued efforts to establish design criteria to prepare conjugates with maximum antigen-binding activity that will enhance the performance of biofunctional nanomaterials.

In Vitro Evaluation of Cocoa Pod Husk Pectin as a Carrier for Chronodelivery of Hydrocortisone Intended for Adrenal Insufficiency.

This study evaluated the in vitro potential of cocoa pod husk (CPH) pectin as a carrier for chronodelivery of hydrocortisone intended for adrenal insufficiency. FTIR studies found no drug-CPH pectin interactions, and chemometric analysis showed that pure hydrocortisone bears closer similarity to hydrocortisone in hot water soluble pectin (HWSP) than hydrocortisone in citric acid soluble pectin (CASP). CPH pectin-based hydrocortisone matrix tablets (~300 mg) were prepared by direct compression and wet granulation techniques, and the tablet cores were film-coated with a 15% HPMC formulation for timed release, followed by a 12.5% Eudragit® S100 formulation for acid resistance. In vitro drug release studies of the uncoated and coated matrix tablets in simulated gastrointestinal conditions showed that wet granulation tablets exhibit greater retardation of drug release in aqueous medium than directly compressed tablets. CASP showed greater suppression of drug release in aqueous medium than HWSP. Wet granulation HWSP-based matrix tablets coated to a final coat weight gain of ~25% w/w were optimized for chronodelivery of hydrocortisone in the colon. The optimized tablets exhibited a lag phase of ~6 h followed by accelerated drug release in the colonic region and have potential to control night time cortisol levels in patients with adrenal insufficiency.