Effects of chain length on oligopeptide hydrogelation

The co-assembly of mutually complementary, but self-repulsive oligopeptide pairs into viscoelastic hydrogels has been studied. Oligopeptides of 6, 10, and 14 amino acid residues were used to investigate the effects of peptide chain length on the structural and mechanical properties of the resulting hydrogels. Biophysical characterizations, including dynamic rheometry, small-angle X-ray scattering (SAXS) and fluorescence spectroscopy, were used to investigate hydrogelation at the bulk, fiber, and molecular levels, respectively. Upon mixing, the 10-mer peptides and the 14-mer peptides both form hydrogels while the 6-mer peptides do not. SAXS studies point to morphological similarity of the cross-sections of fibers underlying the 10:10 and 14:14 gels. However, fluorescence spectroscopy data suggest tighter packing of the amino acid side chains in the 10:10 fibers. Consistent with this tighter packing, dynamic rheometry data show that the 10:10 gel has much higher elastic modulus than the 14:14 mer (18 kPa vs. 0.1 kPa). Therefore, from the standpoint of mechanical strength, the optimum peptide chain length for this class of oligopeptide-based hydrogels is around 10 amino acid residues.     

Adhesion and structure properties of protein nanomaterials containing hydrophobic and charged amino acids

Protein polymers are being used or considered for biobased adhesives and coating materials. Most adhesives derived from macro protein molecules work through receptors or cross-links to bring about adhesion. The adhesion mechanism of protein polymers would lead to better understanding of adhesives and the discovery of new practical properties of protein polymers at both nano- and macro-scales. The objective of this research work was to study adhesion properties of protein polymers at nanoscale (a peptide adhesive with nanometer-scale units that range in size of several nanometers, defined as protein nanomaterial). Seven protein nanomaterial samples with different degrees of adhesive strength were designed and synthesized using solid phase chemistries. All protein nanomaterials contain a common hydrophobic core flanked by charged amino acid sequences. The adhesion properties of the protein nanomaterials were investigated at different pH values and curing temperatures. The protein nanomaterials self aggregate and interact with the wood surface. The protein nanomaterial KKK-FLIVIGSII-KKK identified in this study had high adhesive strength toward wood. It had the highest shear strength at pH 12, with an amino acid sequence that was very hydrophobic and uncharged. This protein nanomaterial underwent structural analyses using circular dichroism, laser-Fourier transform infrared, and laser desorption mass spectrometry. At pH 12 this peptide adopted a pH-induced beta-like conformation. Adhesive strength reflects contributions of both hydrogen bonding and van der Waals interactions. Ionic and covalent bonds do not appear to be significant factors for adhesion in this study.     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

Virtual polymorphism: finding divergent peptide matches in mass spectrometry data

The prevailing method of analyzing tandem-MS data for protein identification involves the comparison of peptide molecular weight and fragmentation data to theoretically predicted values, based on known protein sequences in databases. This is generally effective since proteins from most species under study are in the database or have sufficient homology to allow significant matching. We have encountered difficulties identifying proteins from fungal species Alternaria alternata due to significant interspecies protein sequence differences (divergence) and its absence from the database. This common household mold causes asthma and allergy problems, but the genome has not been sequenced. De novo sequencing and error-tolerant methods can facilitate protein identifications in divergent, unsequenced species. But these standard methods can be laborious and only allow single amino acid substitution, respectively. We have developed an alternative approach focusing on database engineering, predicting biologically rational polymorphism using statistically weighted amino acid substitution information held in BLOSUM62. Like other second pass methods, it is based on the initially identified protein. However, this approach allows more control over sequences to be considered, including multiple changes per peptide. The results show considerable improvement for routine protein identification and the potential for rescuing otherwise unconvincing identifications in unusually divergent species.     

Capillary electrophoresis frontal analysis for characterization of alphavbeta3 integrin binding interactions

The specific binding characteristics of alphavbeta3 integrins with an arginine-glycine-aspartic-acid (RGD) containing fluorescently labeled cyclic peptide is investigated with capillary electrophoresis-frontal analysis method. The new algorithm used to calculate the binding constants and binding stoichiometry was derived without the assumptions made in the commonly used Scatchard Plot method, thus enabling the determination of specific binding parameters in the presence of nonspecific binding. The alphavbeta3 integrin, a membrane protein, was studied in solution, without the need of immobilization or any other kind of modification. An RGD containing fluorescently labeled cyclic pentapeptide is used as the ligand with both specific and nonspecific binding characteristics, and an arginine-alanine-aspartic-acid (RAD) containing peptide is used as the control for nonspecific binding. While a typical specific binding isotherm has a shape of a rectangular hyperbola, a nonspecific binding isotherm is linear in the same ligand concentration region. A 1:2 specific binding stoichiometry was revealed with the second binding having a similar affinity compared to the first binding event.     

MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry

The characterization of proteomes by mass spectrometry is largely limited to organisms with sequenced genomes. To identify proteins from organisms with unsequenced genomes, database sequences from related species must be employed for sequence-similarity protein identifications. Peptide sequence tags (Mann, 1994) have been used successfully for the identification of proteins in sequence databases using partially interpreted tandem mass spectra of tryptic peptides. We have extended the ability of sequence tag searching to the identification of proteins whose sequences are yet unknown but are homologous to known database entries. The MultiTag method presented here assigns statistical significance to matches of multiple error-tolerant sequence tags to a database entry and ranks alignments by their significance. The MultiTag approach has the distinct advantage over other sequence-similarity approaches of being able to perform sequence-similarity identifications using only very short (2-4) amino acid residue stretches of peptide sequences, rather than complete peptide sequences deduced by de novo interpretation of tandem mass spectra. This feature facilitates the identification of low abundance proteins, since noisy and low-intensity tandem mass spectra can be utilized.     

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.     

Use of a designed peptide array to infer dissociation trends for nontryptic peptides in quadrupole ion trap and quadrupole time-of-flight mass spectrometry

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.     

Probability-based validation of protein identifications using a modified SEQUEST algorithm

Database-searching algorithms compatible with shotgun proteomics match a peptide tandem mass spectrum to a predicted mass spectrum for an amino acid sequence within a database. SEQUEST is one of the most common software algorithms used for the analysis of peptide tandem mass spectra by using a cross-correlation (XCorr) scoring routine to match tandem mass spectra to model spectra derived from peptide sequences. To assess a match, SEQUEST uses the difference between the first- and second-ranked sequences (ACn). This value is dependent on the database size, search parameters, and sequence homologies. In this report, we demonstrate the use of a scoring routine (SEQUEST-NORM) that normalizes XCorr values to be independent of peptide size and the database used to perform the search. This new scoring routine is used to objectively calculate the percent confidence of protein identifications and posttranslational modifications based solely on the XCorr value.     

Engineered allosteric ribozymes that sense the bacterial second messenger cyclic diguanosyl 5'-monophosphate

A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-5'-monophosphate (c-di-GMP) have been created by using in vitro selection. An RNA library was generated by using random-sequence bridges to join a hammerhead self-cleaving ribozyme to an aptamer from a natural c-di-GMP riboswitch. Specific bridge sequences, called communication modules, emerged through two in vitro selection efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptamer. Representative RNAs were found that exhibit EC(50) (half-maximal effective concentration) values for c-di-GMP as low as 90 nM and IC(50) (half-maximal inhibitory concentration) values as low as 180 nM. The allosteric RNAs display molecular recognition characteristics that mimic the high discriminatory ability of the natural aptamer. Some engineered RNAs operate with ribozyme rate constants approaching that of the parent hammerhead ribozyme. By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in three mutant strains of Escherichia coli were quantitatively estimated from cell lysates. Our findings demonstrate that engineered c-di-GMP-sensing ribozymes can be used as convenient tools to monitor c-di-GMP levels from complex biological or chemical samples. Moreover, these ribozymes could be employed in high-throughput screens to identify compounds that trigger c-di-GMP riboswitch function.     

Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.     

Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met > Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.     

茶尺蠖普通气味结合蛋白基因片段cDNA的克隆与序列分析

根据同源性设计简并性引物,采用RT-PCR方法扩增出了茶尺蠖普通气味结合蛋白GOBP1和GOBP2的基因cDNA片段,大小分别为415 bp和352 bp.测序结果在NCBI中经BLAST搜索,结果表明,两者与已报道的鳞翅目昆虫的气味结合蛋白基因片段或全长的同源性分别为79%~83%和78%~90%.根据两个核苷酸片段推导出的氨基酸残基数分别为138个和117个,均具有6个保守的半胱氨酸位点,符合典型的气味结合蛋白的特点.经BLAST搜索,与其他鳞翅目昆虫GOBP1和GOBP2氨基酸序列的同源性分别为62%~82%和76%~88%.     

苏云金芽孢杆菌营养期杀虫蛋白基因vip3A的研究

根据已知vip3A基因序列设计一对特异性引物VIP3／VIP5,对218株Bt菌株进行PCR鉴定,有51株含有vip3A类基因,其中12株为不同亚种的标准菌株,有4株标准菌株不含有该基因。从Bt C9菌株中分离克隆了vip—C9基因,并插入表达载体pET—21b,转化大肠杆菌BL21,诱导表达出分子量88．6kDa的可溶性蛋白,表达产物对甜菜夜蛾和棉铃虫具有较高的杀虫活性,LC50分别为0．42ng／mg和25．95ng／mg,对小菜蛾活性较低。构建了缺失蛋白Vip—C9—N(N端去除39个氨基酸组成的信号肽序列),杀虫活性测定结果表明其对甜菜夜蛾的活性显著降低,说明N端39个氨基酸对甜菜夜蛾的活性是必需的。     

Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

Identifying proteins and their modification states and with known levels of confidence remains as a significant challenge for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and amino acid residue sequencing using high-accuracy MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3 132 confident peptide identifications ( approximately 5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3 359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed approximately 5% of the 3 359 identifications to be incorrect and many more as potentially ambiguous (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including nontryptic peptides, peptides with a variety of expected/unexpected chemical modifications, known/unknown post-translational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.     

Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry

Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.     

Derivatization for LC-electrospray ionization-MS: a tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides

We have developed a method for analyzing polar compounds by reversed-phase LC-ESI-MS following esterification of the analytes' free hydroxyl groups with propionyl or benzoyl acid anhydride. The method was applied to members of the plant hormone group cytokinins, which includes adenine bases, ribosides/glycosides, and nucleotides substituted at N-6 with an isoprenoid side chain, spanning a wide range of polarity. It was also used to analyze other compounds of biological importance, e.g., the nucleotides AMP, ADP, and ATP. The formation of more hydrophobic derivatives had a significant impact on two aspects of the analysis. The retention on a reversed-phase material was greatly increased without the use of any acetate/formate buffer or ion pairing reagent, and the ESI response was enhanced, due to the higher surface activities of the derivatives. Detection limits of propionylated cytokinins were in the high-attomole to low-femtomole range, an improvement by factors of 10-100 compared to previously reported figures. Using an automated SPE-based purification method, 12 endogenous cytokinins were quantified in extracts from 20- to 100-mg samples of leaves (from the plant Arabidopsis thaliana) with high accuracy and precision. Furthermore, the chromatographic properties of the benzoylated AMP, ADP, and ATP in the reversed-phase LC-MS system were much better in terms of retention, separation, and sensitivity than those of their underivatized counterparts, even without the use of any ion pairing reagent. Our data show that derivatization followed by LC-ESI-MS is an effective strategy for analyzing low molecular weight compounds, enabling compounds with a wide range of polarity to be determined in a single-injection LC-MS analysis.     

Assay principle for modulators of protein-protein interactions and its application to non-ATP-competitive ligands targeting protein kinase A

Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.