Selective enrichment of endogenous peptides by chemically modified porous nanoparticles for peptidome analysis

We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8nm) and high-surface area (i.e. 751m(2)/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.     

PET Imaging of the Neuropeptide Y System: A Systematic Review

Neuropeptide Y (NPY) is a vastly studied biological peptide with numerous physiological functions that activate the NPY receptor family (Y₁, Y₂, Y₄ and Y₅). Moreover, these receptors are correlated with the pathophysiology of several diseases such as feeding disorders, anxiety, metabolic diseases, neurodegenerative diseases, some types of cancers and others. In order to deepen the knowledge of NPY receptors’ functions and molecular mechanisms, neuroimaging techniques such as positron emission tomography (PET) have been used. The development of new radiotracers for the different NPY receptors and their subsequent PET studies have led to significant insights into molecular mechanisms involving NPY receptors. This article provides a systematic review of the imaging biomarkers that have been developed as PET tracers in order to study the NPY receptor family.     

Design of peptide–dendrimer conjugates with tumor homing and antitumor effects

Development of drug delivery systems for cancer therapy is a crucial issue. Previously, some peptides were designed as tumor homing cell-penetrating peptides with antitumor activities. In this study, dual function dendrimers with tumor targeting activities and antitumor effects were designed using the tumor targeting CPP44 peptide for acute myelogenous leukemia (AML) and the antitumor p16^INK4a peptide. Two types of peptide–dendrimer conjugates were synthesized. One was a CPP44-linked p16^INK4a peptide-conjugated dendrimer (tandem linked dendrimer) and the other was a dendrimer conjugated with separate CPP44 and p16^INK4a peptides (parallel linked dendrimer). In addition, a peptide cathepsin B substrate was linked to the antitumor p16^INK4a peptide to release it from the carriers. These peptide–dendrimer conjugates produced more effective antitumor effects than a CPP44-linked p16^INK4a peptide. The parallel linked dendrimer showed less association with AML cells than the tandem linked dendrimer, but had greater antitumor effects. This suggested that both cellular uptake and antitumor peptide cleavage affected the antitumor activities of dual functional peptide-conjugated dendrimers.     

Highly improved dielectric behaviour of ferronematic nanocomposite for display application

We report the investigation of influence of nickel zinc ferrite magnetic nanoparticles (NZFO (Ni_0.5Zn_0.5Fe₂O₄)) on phase transition, optical and dielectric properties in a nematic liquid crystal (NLC). The interaction of NZFO nanoparticles with NLC was confirmed by the formation of ferronematic droplets due to the transfer of magnetic orientational effect onto the underlying NLC matrix. The doping results in shift of nematic to isotropic transition to low-temperature region. An enhancement in the value of refractive index is observed in the nematic region after the addition of NZFO nanoparticles. The dielectric constant of NLC was remarkably enhanced by 10 times after doping, which is found to be maximum at 0.1 wt% concentration of NZFO nanoparticles. The decrease in the value of dissipation factor in low-frequency region shows that the magnetic nanoparticles are able to trap ionic impurities effectively. The obtained results suggest that the optimum amount of doping concentration is 0.1 wt% of NZFO nanoparticles in NLC due to high dielectric constant with low dissipation factor and high refractive index with high dispersive power at room temperature.     

Thermal and electro-optical properties of cerium-oxide-doped liquid-crystal devices

Doping with cerium (IV) oxide (CeO₂) nanoparticles can significantly enhance the thermal stability and electro-optical (EO) properties of nematic liquid crystal (NLC) systems. Thermal stability was improved without aggregation and reduction of transmittance by adding CeO₂ nanoparticles in a liquid crystal medium. In particular, the EO properties greatly improved in CeO₂-dispersed NLC cells. The threshold voltage reduced from 3.027 to 2.279 V and the response time decreased from 13.097 to 9.970 ms with increased CeO₂ nanoparticles in the NLC cells. The improved properties of liquid crystals doped with CeO₂ nanoparticles depend on the anchoring energy and the electric field of the CeO₂-dispersed liquid crystal displays.     

Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption

Control over selective recognition of biomolecules on inorganic nanoparticles is a major challenge for the synthesis of new catalysts, functional carriers for therapeutics, and assembly of renewable biobased materials. We found low sequence similarity among sequences of peptides strongly attracted to amorphous silica nanoparticles of various size (15-450 nm) using combinatorial phage display methods. Characterization of the surface by acid base titrations and zeta potential measurements revealed that the acidity of the silica particles increased with larger particle size, corresponding to between 5% and 20% ionization of silanol groups at pH 7. The wide range of surface ionization results in the attraction of increasingly basic peptides to increasingly acidic nanoparticles, along with major changes in the aqueous interfacial layer as seen in molecular dynamics simulation. We identified the mechanism of peptide adsorption using binding assays, zeta potential measurements, IR spectra, and molecular simulations of the purified peptides (without phage) in contact with uniformly sized silica particles. Positively charged peptides are strongly attracted to anionic silica surfaces by ion pairing of protonated N-termini, Lys side chains, and Arg side chains with negatively charged siloxide groups. Further, attraction of the peptides to the surface involves hydrogen bonds between polar groups in the peptide with silanol and siloxide groups on the silica surface, as well as ion-dipole, dipole-dipole, and van-der-Waals interactions. Electrostatic attraction between peptides and particle surfaces is supported by neutralization of zeta potentials, an inverse correlation between the required peptide concentration for measurable adsorption and the peptide pI, and proximity of cationic groups to the surface in the computation. The importance of hydrogen bonds and polar interactions is supported by adsorption of noncationic peptides containing Ser, His, and Asp residues, including the formation of multilayers. We also demonstrate tuning of interfacial interactions using mutant peptides with an excellent correlation between adsorption measurements, zeta potentials, computed adsorption energies, and the proposed binding mechanism. Follow-on questions about the relation between peptide adsorption on silica nanoparticles and mineralization of silica from peptide-stabilized precursors are raised.     

Cell-Penetrating Peptides: Correlation between Peptide-Lipid Interaction and Penetration Efficiency

Cell-penetrating peptides are used in the delivery of peptides and biologics, with some cell-penetrating peptides found to be more efficient than others. The exact mechanism of how they interact with the cell membrane and penetrate it, however, remains unclear. This study attempts to investigate the difference in free energy profiles of three cell-penetrating peptides (TAT, CPP1 and CPP9) with a model lipid bilayer (DOPC) using molecular dynamics pulling simulations with umbrella sampling. Potential mean force (PMF) and free energy barrier between the peptides and DOPC are determined using WHAM analysis and MM-PBSA analysis, respectively. CPP9 is found to have the smallest PMF value, followed by CPP1 and TAT, consistent with the experimental data. YDEGE peptide, however, does not give the highest PMF value, although it is a non-cell-permeable peptide. YDEGE is also found to form water pores, alongside with TAT and CPP9, suggesting that it is difficult to distinguish true water pore formation from artefacts arising from pulling simulations. On the contrary, free energy analysis of the peptide-DOPC complex at the lipid-water interface with MM-PBSA provides results consistent with experimental data with CPP9 having the least interaction with DOPC and lowest free energy barrier, followed by CPP1, TAT and YDEGE. These findings suggest that peptide-lipid interaction at the lipid-water interface has a direct correlation with the penetration efficiency of peptides across the lipid bilayer.     

Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G‐protein‐coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double‐cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G‐protein‐coupled Y₂ receptor (Y₂R). Solid‐state NMR measurements of specific isotope‐labeled NPY in complex with in vitro folded Y₂R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C‐terminal α‐helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T³² to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y₂R.     

Improvement of the electro-optical properties of nematic liquid crystals doped with strontium titanate nanoparticles at various doping concentrations

ABSTRACT

In this study, we doped homogenous aligned nematic liquid crystal (NLC) systems with strontium titanate (SrTiO₃) nanoparticles (NPs), and investigated the impact of doping concentration on the NLC’s electro-optical (EO) properties. SrTiO₃ NP-doped NLC cells maintained a high optical transmittance of 77.51% to 78.41% compared to pure NLC cells (78.09%). At a 0.1 wt.% SrTiO₃ NP doping concentration, twisted-nematic (TN)-LC cells exhibited enhanced EO performance, with a reduced threshold voltage from 1.70V to 1.61V and a shorter response time from 17.03 ms to 10.66 ms without optical defects and degradations. We also observed an improvement in thermal endurance for doping concentrations of 0.05 and 0.1 wt.%.     

Fast removal and determination of doxycycline in water samples and honey by Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> magnetic nanoparticles

Adsorption characteristics and doxycycline (DC) removal efficiency of Fe₃O₄ magnetic nanoparticles as adsorbents have been determined by investigating the effects of pH, concentration of the DC, amount of adsorbents, contact time, ionic strength and temperature. The mechanism of adsorption was also studied. The adsorption of DC to the Fe₃O₄ magnetic nanoparticles could be described by Langmuir-type adsorption isotherms. Short contact time between the reagents, reusability of Fe₃O₄ for three times after recycling of the nanoparticles, good precision and accuracy, wide working pH range and high breakthrough volume are among the highlights of this procedure. The proposed extraction and determination procedure based on magnetic nanoparticles as adsorbent was successfully applied to the determination of DC spiked in honey and various water samples. The method presented here is fast, simple, cheap and robust, and it does not require the use of organic solvents. Also, the method needs only a magnet and can be performed in any laboratory without sophisticated equipment.     

Lactosylated casein phosphopeptides as specific indicators of heated milks

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α_s2-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.     

Interaction of the neurotransmitter, neuropeptide Y, with phospholipid membranes: film balance and fluorescence microscopy studies

The association of neuropeptide Y (NPY) with air-water interfaces and with phospholipid monolayers on water subphases and on physiological buffer has been investigated. Surface pressure (pi) versus molecular area (A) relations of the peptide at water surfaces depend on the concentration of the spreading solutions. Independent of that concentration, they show a transition from a low-density state to a high-density state at pi approximately 12 mN/m. Similar features are observed in the NPY adsorption to preformed monolayers (Deltapi(t --> infinity) as a function of pii = pi (t = 0) where t = 0 signifies the time of peptide injection). The transition is also observed in cospread lipid-NPY monolayers and is interpreted as the exclusion of the peptide from the surface layer. The reproducibility of the isotherms after expansion suggests that cospread lipid-peptide monolayers are thermodynamically stable and that the peptide remains associated with the monolayer after exclusion from the lipid surface. A comparison of NPY association with zwitterionic and with anionic lipids as well as a comparison of the interactions on pure water and on physiological buffer suggest that electrostatic attraction plays a major role in the energetics of peptide binding to the membrane surface. Dual label fluorescence microscopy demonstrates that the peptide associates preferentially with the disordered, liquid condensed monolayer phase and also suggests that it self-aggregates upon exceeding a critical surface concentration. A NPY variant with a distorted alpha-helix interacts with the surface as strongly as the natural NPY but expands the monolayers more. This suggests that the helix motif in the peptide is more important for the interaction with the receptor than for binding of the peptide to the membrane surface. In context, these observations attribute a specific role to the membrane in funneling the signal peptide to its membrane receptor.     

Pyridone dipeptide backbone scan to elucidate structural properties of a flexible peptide segment

Whereas the C-terminal fragment of neuropeptide Y (NPY) has been structurally well-defined both in solution and as membrane-bound, detailed structural information regarding the proline-rich N-terminus is still missing. The systematic variation of each position by a conformationally constrained pyridone dipeptide building block within the amino terminal segment of NPY leads to a systematic receptor subtype selectivity of the neuropeptide. Thereby, the systematic dipeptide scan proved superior to the traditional L-Ala scan because it showed how to modify the N-terminus in order to obtain increasingly more Y1 or Y5 receptor selective ligands. NMR and CD spectroscopic analyses were used to characterize the stepwise rigidification of the N-terminus of NPY when up to three dipeptide building blocks were incorporated by solid-phase peptide synthesis. The pyridone dipeptide increases the hydrophobicity of the amino terminus of NPY, and this allows the tuning of the membrane affinity of NPY. The amphiphilic C-terminal helix of 3-fold-substituted NPY thus becomes visible by selective line broadening in the (1)H NMR. Accordingly, we could structurally characterize protein segments that are too flexible for other methods.     

Analysis of electro-optical and dielectric parameters of TiO2 nanoparticles dispersed nematic liquid crystal

The present investigation is focused on to find out the role of TiO₂ nanoparticles (NPs) on altering the dielectric and electro-optical parameters of nematic liquid crystal (NLC). In addition to this, we also optimized the concentration of dopant (0.25 wt%) for a saturation value of permittivity and dielectric anisotropy in the doped system. Dielectric spectroscopy has been performed with the variation of frequency and temperature to investigate the various dielectric parameters, which demonstrate that the investigated NLC is of positive dielectric anisotropy; the observed result shows a decrement in the value of relative permittivity and dielectric anisotropy; however, the permittivity value increases for higher concentration of dopant but remains less than that of pure NLC. Electro-optical measurements have also been performed to compute the optical response of pure and dispersed NLC. It is found that optical response decreases for the NP-doped systems. This optimized concentration of NPs in NLC matrix can have various credential applications in the field of active matrix display and holography.     

Comparison of solvation-effect methods for the simulation of peptide interactions with a hydrophobic surface

In this study we investigated the interaction behavior between thirteen different small peptides and a hydrophobic surface using three progressively more complex methods of representing solvation effects: a united-atom implicit solvation method [CHARMM 19 force field (C19) with Analytical Continuum Electrostatics (ACE)], an all-atom implicit solvation method (C22 with GBMV), and an all-atom explicit solvation method (C22 with TIP3P). The adsorption behavior of each peptide was characterized by the calculation of the potential of mean force as a function of peptide-surface separation distance. The results from the C22/TIP3P model suggest that hydrophobic peptides exhibit relatively strong adsorption behavior, polar and positively-charged peptides exhibit negligible to relatively weak favorable interactions with the surface, and negatively-charged peptides strongly resist adsorption. Compared to the TIP3P model, the ACE and GBMV implicit solvent models predict much stronger attractions for the hydrophobic peptides as well as stronger repulsions for the negatively-charged peptides on the CH(3)-SAM surface. These comparisons provide a basis from which each of these implicit solvation methods may be reparameterized to provide closer agreement with explicitly represented solvation in simulations of peptide and protein adsorption to functionalized surfaces.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

基于超高效液相色谱-质谱法的肽段分析中非特异性吸附评估及通用型最小化策略北大核心CSCD

蛋白质组学技术在多肽和蛋白质类新型治疗药物的开发、临床诊断生物标志物的深入发掘中应用广泛。然而,多肽和蛋白质类大分子的非特异性吸附性质给分析方法的开发带来极大挑战,亟须一种通用型的策略去评估和降低非特异吸附对超高效液相色谱-质谱(UPLC-MS)大分子检测造成的负面影响。研究以牛血清白蛋白(BSA)为模型,探讨其酶解后多肽组理化性质与吸附程度之间的相关性;根据肽段的响应和吸附程度设计分级策略;针对高响应、强吸附的ClassⅡ类肽段,从样品制备中离心管、进样瓶的选择,乃至液相色谱系统中色谱柱固定相、流速、梯度、柱温、洗针液的选择全过程设计试验,探讨非特异吸附的影响因素及其通用型最小化策略。结果显示,肽段的被吸附程度与其理化参数HPLC指数(HPLC Index)、肽段长度等显著相关(p<0.05),但仅凭上述参数仅能解释30%肽段的被吸附程度。改性的聚丙烯材料可使肽段溶液在储存或前处理过程中获得较高的回收率(24 h内回收率大于80%)。在对液相色谱条件的考察和优化过程中发现,C_(8)填料的色谱柱、高流速、缓梯度以及强洗针液,可使残留量降至最低(降低为原来的1/150)。柱温对残留的影响在肽段间存在较大个体差异,需要对不同的肽段具体分析以得到较少量的残留。研究以详实的数据考察并最小化模型肽段组在分析过程中的非特异吸附,提示了蛋白质类大分子药物分析方法建立中应重点关注的影响因素及其有效的解决方案。     

Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTipPPY-C18) based on hybrid electrostatic, Π–Π stacking and hydrophobic interactions for mass spectrometric analysis

Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and Π–Π stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip_PPY-C18 can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip_PPY-C18 should be able to facilitate the exploration of large scale phosphoproteome.