Elastin-Hyaluronan Bioconjugate as Bioactive Component in Electrospun Scaffolds

Hyaluronic acid or hyaluronan (HA) and elastin-inspired peptides (EL) have been widely recognized as bioinspired materials useful in biomedical applications. The aim of the present work is the production of electrospun scaffolds as wound dressing materials which would benefit from synergic action of the bioactivity of elastin peptides and the regenerative properties of hyaluronic acid. Taking advantage of thiol-ene chemistry, a bioactive elastin peptide was successfully conjugated to methacrylated hyaluronic acid (MAHA) and electrospun together with poly-D,L -lactide (PDLLA). To the best of our knowledge, limited reports on peptide-conjugated hyaluronic acid were described in literature, and none of these was employed for the production of electrospun scaffolds. The conformational studies carried out by Circular Dichroism (CD) on the bioconjugated compound confirmed the preservation of secondary structure of the peptide after conjugation while Scanning Electron Microscopy (SEM) revealed the supramolecular structure of the electrospun scaffolds. Overall, the study demonstrates that the bioconjugation of hyaluronic acid with the elastin peptide improved the electrospinning processability with improved characteristics in terms of morphology of the final scaffolds.     

Separation of small molecular peptides with the same amino acid composition but different sequences by high performance liquid chromatography-electrospray ionization-mass spectrometry

Peptidomics has emerged as a new discipline in recent years. Mass spectrometry (MS) is the most universal and efficient tool for structure identification of proteins and peptides. However, there is a limitation for the identification of peptides with the same amino acid composition but different sequences because these peptides have identical mass spectra of molecular ions. This paper presents a high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for the separation of small molecular peptides with the same amino acid composition but different sequences. Two tripeptides of Gly-Ser-Phe and Gly-Phe-Ser were used as a model sample. The separation behavior has been investigated and the separation conditions have been optimized. Under the optimum conditions, good repeatability was achieved. The developed method could provide a helpful reference for the separation of other peptides with the same amino acid composition but different sequences in the study of proteomics and peptidomics.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Solid Phase Peptide Synthesis

The last two decades have been an era of rapid progress in peptide research. This era was begun by the work of Sanger on the amino acid sequence determination of insulin and by du Vigneaud on the structure determination and synthesis of oxytocin. This period has seen impressive progress in the structure elucidation and synthesis of many peptides of natural origin and of great biological significance, as well as in methods for sequence determination and chemical synthesis of peptides [1–4]. Perfection of techniques and instruments for automatic determination of the amino acid sequence of peptides and proteins has made possible a greatly broadened understanding of genetics and evolution as well as the more chemical areas of mechanism of action of enzymes and hormones, and physical chemistry of peptides and proteins. Effective methods of peptide synthesis are crucial to progress in this area, because only by synthesis can adequate amounts of important peptides be made available for chemical, biological, and physical studies, as well as for exploration of the structure-function aspects of biological molecules. In general, progress in peptide synthesis has lagged far behind that in amino acid sequence determination. This is not surprising since effective peptide synthesis requires a very sophisticated system of selectively removable protecting groups for functions of the amino acids involved, and the synthesis of a large heteropolytner of defined sequence requires near perfection of each one of the many steps of the assembly. The classical approach to peptide synthesis, using standard organic chemical methods of synthesis and purification of intermediates, has yielded impressive results during these two decades. However, the special problems associated with the assembly of large molecules make staggering investments in time and materials necessary for the synthesis of large peptides or proteins by classical methods.     

Use of a proteomics approach to identify favourable conditions for production of good quality lambskin leather

It is necessary to understand the changes that occur during the initial processing of lamb skins, because these will affect the final quality of the leather. The types of collagen, their macro and micro structures, the presence of proteins other than collagens, and the quantity and the type of proteoglycans, all have a profound effect on the quality of leather. Proteins isolated from untreated or raw sheep skin and from pickled skin (skins treated with sodium sulfide and lime followed by bating with enzymes, then preserved in sodium chloride and sulfuric acid) were significantly different when analysed by use of 2D gel electrophoresis and mass spectrometry. Agarose gel electrophoresis with a very sensitive sequential staining procedure has been used to identify the glycosaminoglycans present in raw and treated skin and their impact on quality of leather. Results showed that effective removal of proteoglycans acting as inter-fibrillar adhesives of collagen fibrils seemed to improve leather quality. Removal of these molecules not only opens up the fibre structure of the skin but may also be important in wool removal. The presence of elastin, which imparts elastic properties to skin, is of significant importance to tanners. The amino acids desmosine and isodesmosine, found exclusively in elastin, were quantitatively analysed to assess the role of elastin in leather quality.     

氨基酸组成相同序列不同的小分子多肽的液相色谱-质谱联用分析

多肽组学是近年来兴起的一门新型学科,质谱已成为多肽组学研究的强有力手段．然而,用于检测具有相同氨基酸组成但序列不同的多肽时,只能给出等同的分子离子峰,在多肽结构解析上受到一定限制．因此,发展色谱分离．质谱检测联用技术是分析具有相同氨基酸组成但序列不同的多肽的有效途径．本文建立了一种氨基酸组成相同序列不同的小分子多肽的反相液相色谱分离-电喷雾离子化质谱检测新方法．该方法采用高效液相色谱-质谱联用技术,以两种三肽Gly．Ser．Phe和Gly．Phe．Ser为模式样品对象,考察了小分子多肽在不同流动相组成、流动相添加剂及pH等条件下的液相色谱行为,并讨论其保留机理．研究结果表明,在最优化的实验条件下,该方法稳定性好,重现性高,为多肽组学研究中的多肽解析提供科学的分析方法．     

Coordination properties of zinc finger peptides revisited: ligand competition studies reveal higher affinities for zinc and cobalt

Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.     

3D-Epitope-Explorer (3DEX): localization of conformational epitopes within three-dimensional structures of proteins

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these "mimotopes" within the three-dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D-Epitope-Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of C(alpha)- or C(beta)-atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV-positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV-1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

On the relation between polypeptide structure and spredptogenic activity. 1. Peptides of cystine

The authors propose for the characterization of strepogenic substances of known composition the specific activity, i. e. the number of WOOLLEY units per μmole. Starting from L -leucyl-L -cystinyl-L -leucyl-L -valyl-L -glutamic acid (of very high strepogenine activity, 400 WOOLLEY units per mg, 230 WOOLLEY units per μmole) seven peptides have been synthesized by suppression and/or replacement of amino acid residues. A study of the relation between activity and structure of these peptides shows that:

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A versatile set of aminooxy amino acids for the synthesis of neoglycopeptides

Four N-alkylaminooxy amino acids have been synthesized in 22-56% overall yield from readily available amino acid precursors. Each amino acid can be efficiently incorporated into peptides using Boc-chemistry-based solid-phase peptide synthesis, and in three of the four cases the resulting peptides can be chemoselectively glycosylated at the aminooxy side chains to generate neoglycopeptides. The range of N-alkylaminooxy amino acids prepared allows attachment of sugars at two-, three-, or four-atom distances from the peptide backbone, and each ensures that attached sugars adopt cyclic conformations. These derivatives provide convenient access to arrays of biologically relevant neoglycopeptides that may be used to probe the influence of attached sugars on the structure and function of peptides and proteins.     

Photoionisation mass spectra of volatile derivatives of short peptides and appearance potentials of their characteristic ions

Mass spectra of the volatile derivatives of short peptides were studied by the photoionisation method with the use of monochromatic photons. The dependence of the intensity of the main peaks on the photon energy was analysed from 7·5 to 13·0 eV. The data obtain reveal the influence of the chemical structure of amino acid residues on the relative probability of the decomposition of peptide molecular ions at the CH? CO and CO? NH bonds, resulting in the formation of positively charged aldimine and amino acid N-terminal fragments, respectively. These data may be used as a basis for the application of the photoionisation technique to mass spectrometric sequential studies in peptides. In peptides containing residues of aliphatic amino acid the decomposition results mainly in the formation of aldimine ions, the stability of which increase with the increase of the alkyl chain size. In peptides containing residues of aromatic amino acids the decomposition is usually observed leading to formation of the amino acid ions. Ionisation potentials, as well as photoionisation efficiency curves and appearance potentials were determined for characteristic ions. Comparison was made of the values of the appearance potentials of different fragments formed upon decomposition of molecular ions. It has been shown that for peptides containing aliphatic amino acid moieties the appearance potentials of aldimine fragments are always lower than those inherent to peptides containing aromatic amino acid residues. The larger the size of an aliphatic chain, the lower is the energy of formation of these fragments. For all the compounds studied, including the peptides containing aromatic amino acid residues, the appearance potentials of the aldimine ions did not exceed those of the amino acid ions. These data indicate that, contrary to the experiments with electron-impact with energy of about 70 eV, upon ionisation with photons with energy from 7·5 to 13·0 eV, the aldimine fragments appear directly due to primary decomposition of molecular ions, independent of the formation of the amino acid fragments. The photoionisation efficiency curves for peptides containing different types of amino acid residues facilitate the choice of an optimal photon energy providing unequivocal information on the amino acid sequence in the peptide under study.     

Changes in Protein Composition in Erythrocyte Membrane of Ethanol—Poisoned Rats After Administration of Teas

Selective hydrolysis of the protein to peptides, their resolution by chromatography and determination of the amino-acid sequence in individual peptides is one method applied to studies of their modifications and mutations in pathological conditions. The purpose of this work is the determination of the green and black tea effect on peptides and their amino acid contents in integral erythrocyte membrane proteins in ethanol intoxicated rats. It was shown that ethanol intoxication causes a decrease in peptides and their amino acid contents in comparison with the control group. The ingestion of green (black) tea with ethanol partially prevents these ethanol-induced changes.     

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as α-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an α-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of α-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure.     

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins.     

Identifying residual structure in intrinsically disordered systems: a 2D IR spectroscopic study of the GVGXPGVG peptide

The peptide amide-I vibration of a proline turn encodes information on the turn structure. In this study, FTIR, two-dimensional IR spectroscopy and molecular dynamics simulations were employed to characterize the varying turn conformations that exist in the GVGX(L)PGVG family of disordered peptides. This analysis revealed that changing the size of the side chain at the X amino acid site from Gly to Ala to Val substantially alters the conformation of the peptide. To quantify this effect, proline peak shifts and intensity changes were compared to a structure-based spectroscopic model. These simulated spectra were used to assign the population of type-II β turns, bulged turns, and irregular β turns for each peptide. Of particular interest was the Val variant commonly found in the protein elastin, which contained a 25% population of irregular β turns containing two peptide hydrogen bonds to the proline C═O.     

Anti-HIV cyclotides

The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.     

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.     

Effects of an arginine-selective tagging procedure on the fragmentation behavior of peptides studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

The presence of arginine as the naturally occurring amino acid with the highest gas-phase basicity strongly influences the fragmentation behavior of peptides undergoing collision-induced dissociation. Using a derivatization procedure recently developed in our group, based on a reversible reaction of the guanidino group with 2,3-butanedione and an arylboronic acid, we examined how this label affects the fragmentation patterns of labeled versus unlabeled peptides in MS/MS experiments. As part of this fundamental study, two groups of model peptides (angiotensins and bradykinins) as well as tryptic peptides were labeled according to our protocol and subjected to collision-induced dissociation (CID) in both a triple quadrupole and a quadrupole ion trap instrument. It was found that for angiotensins containing an AspArg sequence, C-terminal cleavage at Asp that occurs for native peptides was completely inhibited in Arg-labeled peptides. For bradykinins and peptides obtained from tryptic digests of standard proteins, some sample peptides were little affected by the tagging of arginine residues. Others, in contrast, exhibited an almost total loss of nonspecific backbone cleavage and their fragment ion spectra were dominated by loss of the arginine tag. These and other experimental results are discussed in view of the nature of the arginine tag and the concept of proton mobility.