Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.     

Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 1. Behavior of mono- to hexapeptides

For a better understanding of the behavior of solid-phase combinatorial peptide ligands for capturing the red blood cell low-abundance soluble proteome, combinatorial peptides of different lengths from a single amino acid up to a hexapeptide were evaluated. A red blood cell lysate (6 g total protein) was loaded in a cascade fashion to the six columns, which were individually eluted with 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (v/w), and 50 mM citric acid. Each eluate was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional maps, and nanoLC-MS/MS. The results: mixed beads with a single amino acid attached showed the capture of a non-negligible portion of the proteome. A progressive increasing of the length of the peptide bait enlarges the pool of captured proteins. Above a length of four amino acids, a plateau is progressively reached, suggesting that not much could be gained with baits longer than six amino acids. Interestingly, whereas the beads laden with a single amino acid seem to be able to capture large-size proteins (>40 kDa), beads with progressively longer peptides capture additional proteins in the smaller size range (10-50 kDa). This suggests that interactions already begin with a single amino acid, but selectivity requires baits of proper length, at least above four amino acids. Plain beads, with a spacer arm carrying a primary amino terminal group for anchoring the baits, are essentially unable to capture proteins, suggesting that the peptide baits do not act by a mechanism of ion exchange but rather via a complex mixed mode, yielding a specific capture.     

Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of alpha-amino acids.

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of alpha-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing beta-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (omega) of alpha-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The beta-turn, a ubiquitous structural feature formed by two residue (alphaalpha) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramolecular 4-->1 hydrogen bond. Hybrid turns may be classified by comparison with their alphaalpha counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual omega-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of alpha-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all alpha-residue sequences. The use of stereochemically constrained omega-residues promises to expand the range of peptide design strategies to include omega-amino acids. This approach is exemplified by well-folded structures like the C12 (alphagamma) and C14 (gammagamma) helices formed in short peptides containing multiply substituted gamma-residues. The achiral gamma-residue gabapentin is a readily accessible building block in the design of peptides containing gamma-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.     

Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.     

Gas-phase separations of electrosprayed peptide libraries.

High-resolution ion mobility spectrometry has been combined with time-of-flight mass spectrometry for analysis of a combinatorial peptide library that is expected to contain 676 components. In this approach, the components of a mixture of three residue peptides, having the general form (D)Phe-Xxx-Xxx-CONH2 (where Xxx is randomized over 26 residues including 10 naturally occurring amino acids and 16 synthetic forms) were ionized by electrospray ionization. Ion mobility/time-of-flight distributions have been recorded for all ions using a nested drift(flight) time technique. The improvement in resolving power [(t/delta t) = 100-150 for singly charged ions] was illustrated by analysis of a mixture of tryptic digest peptides using high- and low-resolution instruments. The approach allows many components of the library (e.g., structural, sequence, and stereo isomers) that cannot be distinguished by mass spectrometry alone to be resolved. Impurities due to side reactions appear to be minimal, comprising < 10% of the total ion signal. Direct evidence for approximately 60-70% of the expected peptides is found. Variation in ion abundance for different components indicates that there are differences in solution concentrations or ionization efficiencies for the components.     

新型皱纹盘鲍防御素hd-def的cDNA序列分析、基因组克隆及表达研究

从构建的皱纹盘鲍肝肾cDNA文库中筛选到了鲍防御素基因EST.通过序列分析发现该基因的全长cDNA序列编码66个氨基酸残基,其前体由信号肽、前导肽和成熟肽组成.该前体的成熟肽含42个氨基酸(6个Cys),推测分子量为4323Da,等电点为8.02.氨基酸序列同源性分析表明,该多肽与昆虫防御素的相似性较高,最高可达70%.因成熟肽二级结构具有典型的昆虫防御素结构特征,该多肽应属于抗菌肽中的昆虫防御素亚家族,是一种新型抗菌肽,将其命名为鲍防御素hd-def.采用基因组步移法获得了全长4032bp的基因组序列.分析表明,该基因由3个内含子和4个外显子编码组成;3个内含子大小分别为497bp、2357bp和528bp,其中两个内含子存在于编码信号肽的序列中.用鳗弧菌和金黄色葡萄球菌刺激皱纹盘鲍,能诱导hd-def的表达.实验检测了5种组织,发现hd-def基因仅在肝胰腺中表达,具有明显的组织表达特异性;其表达属于诱导型表达,提示该基因可能参与皱纹盘鲍的抗细菌感染.     

Enzymatic activation of a peptide functionalised gold nanoparticle system for prodrug delivery

Gold nanoparticles (GNPs) with a monolayer of peptides were synthesized as a potential tumour activated cancer drug delivery system. The prodrug system was achieved by the attachment of two varying lengths of peptides to GNPs: An 18 amino acid peptide sequence encompassing a shorter fluorescent labelled (coumarin) six amino acid peptide sequence. The longer peptide chain included the sequence D-AFK that is selectively cleavable by the over-expression of proteases in the vicinity of cancer cells. The protease-mediated exposure of the coumarin was demonstrated by the incubation of peptide capped GNPs with adenocarcinomic human alveolar basal epithelial A549 cells and madin-darby bovine kidney epithelial cells. Confocal laser scanning microscopy studies revealed enhanced fluorescence emission intensities in the cancer cell line as compared to the intensity exhibited by the healthy cell line. This work suggests that GNPs functionalised with a cytotoxic agent or fluorophore encapsulated by longer peptide strands may find useful applications for development of GNPs with therapeutic or diagnostic studies.     

Near-infrared spectrometric determination of di- and tripeptides synthesized by a combinatorial solid-phase method

A new method based on near-infrared (NIR) spectrometry and partial least-squares analysis has been developed for the noninvasive and nondestructive determination of the identity and sequences of amino acid residues in di- and tripeptides. The di- and tripeptides were synthesized from six amino acids with similar structures (Gly, Ala, Leu, Met, Phe, Val) on two different polymer beads (bead with and without a linker) using the solid-phase peptide synthetic method. The developed NIR method is capable of determining the identity of sequences of these di- and tripeptides (with and without the Fmoc protecting group) directly on the polymer beads. It can distinguish not only dipeptides from tripeptides but also peptides with very similar structures (e.g., bead-Gly-Ala-Ala, bead-Gly-Ala-Phe, bead-Gly-Ala-Leu, bead-Gly-Ala-Val, and bead-Gly-Ala-Met). More importantly, the method is capable of distinguishing di- and tripeptides with the same amino acid residues but different sequences (e.g., bead-Gly-Leu-Val from bead-Gly-Val-Leu).     

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.     

Conformational analysis of peptides using Monte Carlo simulations combined with the genetic algorithm

In this paper, genetic algorithm (GA) was used to both sample the conformational spaces and thoroughly search the global conformations of peptides. δ-conotoxin PVIA, a peptide of 29 amino acids, was used to test our procedure. The results indicated that this procedure could not only successfully explore a set of the conformational spaces using a hybrid Monte Carlo simulations/GA minimization but also find the global conformations for most of peptides using pure GA minimization.     

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.     

Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens

The preparation and use of a new class of signal amplification label, quantum dot (QD) layer-by-layer (LBL) assembled polystyrene microsphere composite, for amplified ultrasensitive electronic detection of uropathogen-specific DNA sequences is described. The target DNA is sandwiched between the capture probes immobilized on the magnetic beads and the signaling probes conjugated to the QD LBL assembled polystyrene beads. Because of the dramatic signal amplification by the numerous QDs involved in each single DNA binding event, subfemtomolar level detection of uropathogen-specific DNA sequences is achieved, which makes our strategy among the most sensitive electronic approach for nucleic acid-based monitoring of pathogens. Our signal amplified detection scheme could be readily expanded to monitor other important biomolecules (e.g., proteins, peptides, amino acids, cells, etc.) in ultralow levels and thus holds great potential for early diagnosis of disease biomarkers.     

Development and validation of a fluorescence method to follow the build-up of short peptide sequences on solid 2D surfaces

The modification of material surfaces with short peptide sequences has become an essential step in many biotechnological and biomedical applications. Due to their simple architecture compared to more complex 3D substrates, 2D surfaces are of particular interest for high throughput applications and as model surfaces for dynamic or responsive surface modifications. The decoration of these surfaces with peptides is commonly accomplished by synthesizing the peptide first and subsequently transferring it onto the surface of the substrate. Recently, several procedures have been described for the synthesis of peptides directly onto a 2D surface, thereby simplifying and accelerating the modification of flat surfaces with peptides. However, the wider use of these techniques requires a routine method to monitor the amino acid build-up on the surface. Here, we describe a fast, inexpensive and nondestructive fluorescence based method which is readily accessible to follow the amino acid build-up on solid 2D samples.     

Benzoyl derivatization as a method to improve retention of hydrophilic peptides in tryptic peptide mapping

This study exploits the increase in chromatographic retention that accrues from benzoyl derivatization of primary amines as a tool to increase sequence coverage in tryptic peptide mapping. N-hydroxysuccinamide sulfonyl benzoate quantitatively derivatizes primary amines of peptides. Introduction of the hydrophobic benzoyl moiety into peptides increased retention of peptides during reversed-phase chromatography (RPC), particularly in the case of smaller hydrophilic peptides. Short chain (1-6 amino acids) tryptic fragments of model proteins lysozyme, myoglobin, and cytochrome c derivatized with N-hydroxysuccinamide sulfonyl benzoate eluted in the linear acetonitrile gradient. Application of benzoyl derivatization was further extended to achieve complete sequence coverage of a therapeutic protein, recombinant human growth hormone, and in detection of single amino acid polymorphism.     

Development of an amino acid sequence and D/L-configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate

On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence, whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.     

Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation

We report an online nonenzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissociation tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymatic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two nonenzymatic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids and (ii) the electrochemical-cleavage method is unable to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of standard peptides and proteins as well as an Escherichia coli protein extract.     

Stereoselective determination of amino acids in beta-amyloid peptides and senile plaques

A novel method for the determination of the enantiomeric composition of peptides is presented. In this paper, the focus has been on beta-amyloid peptides from deceased Alzheimer's disease patients. The peptides are hydrolyzed using mineral acid. The free amino acids are derivatized with the chiral reagent (+)- or (-)-1-(9-anthryl)-2-propyl chloroformate and subsequently separated using micellar electrokinetic chromatography (MEKC) and detected using laser-induced fluorescence (LIF) detection. The high separation efficiency of the MEKC-LIF system, yielding approximately 1 million theoretical plates/m for most amino acids, facilitates the simultaneous chiral determination of nine amino acids. The samples that have been analyzed were standard 1-40 beta-amyloid peptides, in vitro precipitated beta-amyloid fibrils, and human senile plaque samples.     

Immobilization of a human epidermal growth factor receptor 2 mimotope-derived synthetic peptide on Au and its potential application for detection of herceptin in human serum by quartz crystal microbalance

Therapeutic antibodies are antigenically similar to human antibodies and are difficult to detect in assays of human serum samples without the use of the therapeutic antibody's complementary antigen. Herein for the first time, we established a platform to detect Herceptin in solutions by using a small (<2.2 kDa), inexpensive, highly stable human epidermal growth factor receptor (HER2) mimotope-derived synthetic peptide immobilized on the surface of a Au quartz electrode. We used the HER2 mimotope as a substitute for the HER2 receptor protein in piezoimmunosensor or quartz crystal microbalance (QCM) assays to detect Herceptin in human serum. We demonstrated that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used to carry out the assays. The detection limit of the piezoimmunosensor assay was 0.038 nM with a linear operating range of 0.038-0.859 nM. Little nonspecific binding to other therapeutic antibodies (Avastin and Rituxan) was observed. Levels of Herceptin in serum samples obtained from treated patients, as ascertained using the synthetic peptide-based QCM assay, were typical for those treated with Herceptin. The findings of this study are significant in that low-cost synthetic peptides could be used in a QCM assay, in lieu of native or recombinant antigens or capture antibodies, to rapidly detect a therapeutic antibody in human serum. The results suggested that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of affinity-based immunosensors to detect a broad range of clinical biomarkers.