Origin of Secondary Structure Transitions and Peptide Self-Assembly Propensity in Trifluoroethanol-Water Mixtures.

Membrane-peptide interactions are key to the formation of helical intermediates in the early stages of amyloidogenesis. Aqueous solutions of 2,2,2-trifluoroethanol (TFE) provide a membrane-mimetic environment capable of promoting and stabilizing local peptide interactions. Uperin 3.5 (U3.5), a 17-residue and amidated antimicrobial peptide, is unstructured in water but self-assembles into fibrils in the presence of salt. Secondary structure transitions linked to U3.5 self-assembly were investigated in TFE/water mixtures, in both the absence and presence of salt, to assess the role of membrane-peptide interactions on peptide self-assembly and amyloid formation. A 5-to-7-fold increase in fibril yield of U3.5 was observed at low TFE concentrations (10% TFE/water v/v) compared with physiological buffer but only in the presence of salt. No aggregation was observed in salt-free TFE/water mixtures. Circular dichroism spectra showed that partial helical structures, initially stabilized by TFE, transitioned to ß-sheet-rich aggregates in a saline buffer. Molecular dynamics simulations confirmed that TFE and salt act synergistically to enhance peptide-peptide interactions, resulting in ß-sheet-rich U3.5 oligomers at low TFE concentrations. Specifically, TFE stabilized amphipathic, helical intermediates, leading to increased peptide-peptide attraction through hydrophobic interactions. The presence of salt further enhanced the peptide-peptide interactions by screening positively charged residues. Thus, the study revealed the role of a membrane mimic in stabilizing helical intermediates on the pathway to amyloid formation in the antimicrobial U3.5 peptide.

Upgrading of the general AMBER force field 2 for fluorinated alcohol biosolvents: A validation for water solutions and melittin solvation.

The standard GAFF2 force field parameterization has been refined for the fluorinated alcohols 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1,1,1,3,3,3-hexafluoropropan-2-one (HFA), which are commonly used to study proteins and peptides in biomimetic media. The structural and dynamic properties of both proteins and peptides are significantly influenced by the biomimetic environment created by the presence of these cosolvents in aqueous solutions. Quantum mechanical calculations on stable conformers were used to parameterize the atomic charges. Different systems, such as pure liquids, aqueous solutions, and systems formed by melittin protein and cosolvent/water solutions, have been used to validate the new models. The calculated macroscopic and structural properties are in agreement with experimental findings, supporting the validity of the newly proposed models.

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent.

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 µs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 µs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.

Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca.

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-[Formula: see text] mixture. The structure is defined by an [Formula: see text]-helix that spans between Ile[Formula: see text]-Ala[Formula: see text], and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its [Formula: see text]-helical conformation between residues Ile[Formula: see text]-Ala[Formula: see text], while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

Changes in the secondary structures and zeta potential of soybean peptide and its calcium complexes in different solution environments.

To illustrate the relationship between environment hydrophobicity and soybean peptide and its calcium complexes when they are absorbed transmembrane, different solution environments (HBS buffer, TFE hydrophobic solution and cell suspension) were used to simulate hydrophilic and hydrophobic environments. In this study, soybean peptides (10-30 kDa) with a high calcium binding capacity were prepared by enzymatic hydrolysis and ultrafiltration. The results of cell experiments showed that the peptide could transport calcium into cells for absorption. Secondary structure changes of the peptide and its calcium complexes in different solution environments showed that the secondary structure of the peptide changed during the transmembrane absorption, and the contents of α-helix and ß-sheet structures increased. Besides, the ß-sheet structures in the peptide-calcium complexes were further converted to an α-helix structure. This conversion may be induced by the hydrophobicity of peptide solutions. In addition, when the conformation changes, the positively charged peptides in the sample will be exposed and then interact with cells, which is beneficial for the transmembrane of peptide-calcium complexes.

Two different regimes in alcohol-induced coil-helix transition: effects of 2,2,2-trifluoroethanol on proteins being either independent of or enhanced by solvent structural fluctuations.

Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifluoroethanol (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by small-angle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol < ethanol ≪ TFE < tert-butanol < 1-propanol, indicating that the inhomogeneity of molecular distribution in the TFE-H2O mixture is unexpectedly comparable to those in the series of mono-ols. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below ∼2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above ∼2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosolvent effects of TFE on proteins/peptides.

Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells.

This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C-N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N-C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.

Direct transfer of tri- and di-fluoroethanol units enabled by radical activation of organosilicon reagents.

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.

Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations.

Proteins of the p53 family are best known for their role in the regulation of cell cycle. The p53 protein, as a model system, has been extensively explored in numerous cancer-related studies. The C-terminal domain (CTD) of p53 is an intrinsically disordered region that gains multiple different conformations at interaction with different binding partners. However, the impact of the surrounding environment on the structural preference of p53-CTD is not known. We investigated the impact of the surrounding environment on the conformational behavior and folding of p53-CTD. Although the entire CTD is predicted as a highly disordered region by several commonly used disorder predictors, based on the secondary structure prediction, we find that a part of the CTD sequence (residues 380-388) is "confused", being predicted to shuffle between the irregular, α-helical and ß-strand structures. First time, we are observing the effect of folding-induced organic solvents, trifluoroethanol and methanol, on the conformation of CTD. Water-miscible organic solvents exert hydrophobic interactions, which are major driving force to trigger structural changes in CTD. By lowering the solution dielectric constant, organic solvents can also strengthen electrostatic interactions. We have also performed Replica Exchange Molecular Dynamic (REMD) simulations for enhanced conformation sampling of the peptide. These simulation studies have also provided detailed insight into the peculiarities of this peptide, explaining its folding behavior in the presence of methanol. We consider that these hydrophobic interactions may have important roles for function-related structural changes of this disordered region.

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0.

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

Inhibiting Human Calcitonin Fibril Formation with Its Most Relevant Aggregation-Resistant Analog.

The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.

About TFE: Old and New Findings.

The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.

Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution.

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and ß-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques - circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.

Immunomodulatory and enhanced antitumor activity of a modified thymosin α1 in melanoma and lung cancer.

Tumor-targeted therapy is an attractive strategy for cancer treatment. Peptide hormone thymosin α1 (Tα1) has been used against several diseases, including cancer, but its activity is pleiotropic. Herein, we designed a fusion protein Tα1-iRGD by introducing the tumor homing peptide iRGD to Tα1. Results show that Tα1-iRGD can promote T-cell activation and CD86 expression, thereby exerting better effect and stronger inhibitory against melanoma and lung cancer, respectively, than Tα1 in vivo. These effects are indicated by the reduced densities of tumor vessels and Tα1-iRGD accumulation in tumors. Moreover, compared with Tα1, Tα1-iRGD can attach more B16F10 and H460 cells and exhibits significantly better immunomodulatory activity in immunosuppression models induced by hydrocortisone. Circular dichroism spectroscopy and structural analysis results revealed that Tα1 and Tα1-iRGD both adopted a helical confirmation in the presence of trifluoroethanol, indicating the structural basis of their functions. These findings highlight the vital function of Tα1-iRGD in tumor-targeted therapy and suggest that Tα1-iRGD is a better antitumor drug than Tα1.

A biophysical insight into the formation of aggregates upon trifluoroethanol induced structural and conformational changes in garlic cystatin.

Intrinsic and extrinsic factors are responsible for the transition of soluble proteins into aggregated form. Trifluoroethanol is among such potent extrinsic factor which facilitates the formation of aggregated structure. It disrupts the interactive forces and destabilizes the native structure of the protein. The present study investigates the effect of trifluoroethanol (TFE) on garlic cystatin. Garlic cystatin was incubated with increasing concentration of TFE (0-90% v/v) for 4 h. Incubation of GPC with TFE induces structural changes thereby resulting in the formation of aggregates. Inactivation of garlic phytocystatin was confirmed by cysteine proteinase inhibitory activity. Garlic cystatin at 30% TFE exhibits native-like secondary structure and high ANS fluorescence, thus suggesting the presence of molten globule state. Circular dichroism and FTIR confirmed the transition of the native alpha-helical structure of garlic cystatin to the beta-sheet structure at 60% TFE. Furthermore, increased ThT fluorescence and redshift in Congo red absorbance assay confirmed the presence of aggregates. Rayleigh and turbidity assay was also performed to validate the aggregation results. Scanning electron microscopy was followed to analyze the morphological changes which confirm the presence of sheath-like structure at 60% TFE. The study sheds light on the conformational behavior of a plant protein when kept under stress condition induced by an extrinsic factor.

Temperature-Induced Phase Separation in Molecular Assembly of Nanotubes Comprising Amphiphilic Polypeptoid with Poly( N-ethyl glycine) in Water by a Hydrophilic-Region-Driven-Type Mechanism.

Two kinds of amphiphilic polypeptoids having different types of hydrophilic polypeptoids, poly(sarcosine)- b-(l-Leu-Aib)6 (ML12) and poly( N-ethyl glycine)- b-(l-Leu-Aib)6 (EL12), were self-assembled via two paths to phase-separated nanotubes. One path was via sticking ML12 nanotubes with EL12 nanotubes and the other was a preparation from a mixture of ML12 and EL12 in solution. In either case, nanotubes showed temperature-induced phase separation along the long axis, which was observed by two methods of labeling one phase with gold nanoparticles and fluorescence resonance energy transfer between the components. The phase separation was ascribed to aggregation of poly( N-ethyl glycine) blocks over the cloud point temperature. The addition of 5% trifluoroethanol was needed for the phase separation because the tight association of the helices in the hydrophobic region should be loosened to allow lateral diffusion of the components to be separated. The phase separation in molecular assemblies in water based on the hydrophilic-region-driven-type mechanism therefore requires sophisticated balances of association forces exerting among the hydrophilic and hydrophobic regions of the amphiphilic polypeptoids.

Structural Stability of Peptidic His-Containing Proton Wire in Solution and in the Adsorbed State.

Molecular wires are functional molecules applicable in the field of transfer processes in technological and biochemical applications. Besides molecular wires with the ability to transfer electrons, research is currently focused on molecular wires with high proton affinity and proton transfer ability. Recently, proposed peptidic proton wires (H wires) are one example. Their ability to mediate the transport of protons from aqueous solutions onto the surface of a Hg electrode in a catalytic hydrogen evolution reaction was investigated by constant-current chronopotentiometric stripping. However, elucidating the structure of H wires and rationalizing their stability are key requirements for their further research and application. In this article, we focus on the His (H) and Ala (A)-containing peptidic H wire A3-(H-A2)6 in solution and after its immobilization onto the electrode surface in the presence of the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE). We found that the solvent containing more than 25% of TFE stabilizes the helical structure of A3-(H-A2)6 not only in solution but also in the adsorbed state. The TFE efficacy to stabilize α-helical structure was confirmed using high-resolution nuclear magnetic resonance, circular dichroism, and molecular dynamics simulation. Experimental and theoretical results indicated A3-(H-A2)6 to be a high proton-affinity peptidic H wire with an α-helical structure stabilized by TFE, which was confirmed in a comparative study with hexahistidine as an example of a peptide with a definitely disordered and random coil structure. The results presented here could be used for further investigation of the peptidic H wires and for the application of electrochemical methods in the research of proton transfer phenomena in general.

Fluoroalcohol - Induced coacervates for selective enrichment and extraction of hydrophobic proteins.

As previously reported, fluoroalcohols can induce coacervation in aqueous solutions of amphiphilic compounds with subsequent formation of two-phase systems, where one phase is enriched in amphiphile and fluoroalcohol and the other is primarily an aqueous - rich phase. This study focuses on the use of simple coacervates made of a single component amphiphile induced by a fluoroalcohol for extraction and enrichment of proteins. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) were used to induce coacervation in the aqueous solutions of a cationic amphiphile, cetyltrimethylammonium bromide (CTAB) or tetra-n-butylammonium bromide (TBAB). Cationic amphiphiles (CTAB, TBAB) formed two-phase coacervate systems in a basic pH and/or sufficient ionic strength depending on the strength of coacervator (HFIP or TFE). The phase diagrams for TBAB paired with HFIP or TFE coacervates were created. By increasing the concentration of coacervator (HFIP or TFE) at a constant surfactant concentration, transition from a single liquid phase to a two or multiple phase mixture, and then eventually to a single liquid phase was observed. TBAB/HFIP mixture without additives showed a unique three-phase system before transitioning to a two-phase system upon increasing HFIP concentration. However, salt addition eliminated this three-phase region and expanded the region of two-phase formation. Select two-phase systems composed of TBAB and a perfluoroalcohol (HFIP or TFE) were utilized to extract model proteins of ranging hydrophobicity. All coacervate phases extracted bacteriorhodopsin, a membrane protein, and gramicidin, a very hydrophobic polypeptide ion channel. The most hydrophilic protein in the mixture, ribonuclease A, remained in aqueous phases. The coacervates formed from TBAB/TFE/200 mM NaCl mixture and TBAB/HFIP mixture exhibited the most selectivity in extracting proteins of high hydrophobicity. The partition coefficient (P) for each protein was calculated using the ratio of the protein concentration in the coacervate to that in the aqueous-rich phases. TBAB (50 mM)/HFIP (8%, v/v) coacervate showed remarkable selectivity and a high partition coefficient (>100) for both bacteriorhodopsin and gramicidin. Thus, this system may potentially be beneficial for facile fractionation of hydrophobic and membrane proteins in proteomics applications.

Enhanced cellular uptake and tumor penetration of nanoparticles by imprinting the "hidden" part of membrane receptors for targeted drug delivery.

The widely existing transmembrane helices can serve as a novel type of binding site for recognizing corresponding membrane receptors. Through imprinting the transmembrane domain of certain receptors, here we report the construction of polymeric nanoparticles which can achieve enhanced cellular uptake and permeability in target tissues for tumor-targeted drug delivery.

Corona Discharge Suppression in Negative Ion Mode Nanoelectrospray Ionization via Trifluoroethanol Addition.

Negative ion mode nanoelectrospray ionization (nESI) is often utilized to analyze acidic compounds, from small molecules to proteins, with mass spectrometry (MS). Under high aqueous solvent conditions, corona discharge is commonly observed at emitter tips, resulting in low ion abundances and reduced nESI needle lifetimes. We have successfully reduced corona discharge in negative ion mode by trace addition of trifluoroethanol (TFE) to aqueous samples. The addition of as little as 0.2% TFE increases aqueous spray stability not only in nESI direct infusion, but also in nanoflow liquid chromatography (nLC)/MS experiments. Negative ion mode spray stability with 0.2% TFE is approximately 6× higher than for strictly aqueous samples. Upon addition of 0.2% TFE to the mobile phase of nLC/MS experiments, tryptic peptide identifications increased from 93 to 111 peptides, resulting in an average protein sequence coverage increase of 18%.