Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.     

Rational design of complementary peptides to the betaAmyloid 29-42 fusion peptide: an application of PepDesign

Peptides in solution currently exist under several conformations; an equilibrium which varies with solvent polarity. Despite or because of this structure versatility, peptides can be selective biological tools: they can adapt to a target, vary conformation with solvents and so on. These capacities are crucial for cargo carriers. One promising way of using peptides in biotechnologies is to decipher their medium-sequence-structure-function relationships and one approach is molecular modelling. Only few "in silico" methods of peptide design are described in the literature. Most are used in support of experimental screening of peptide libraries. However, the way they are made does not teach us much for future researches. In this paper, we describe an "in silico" method (PepDesign) which starts by analysing the native interaction of a peptide with a target molecule in order to define which points are important. From there, a modelling protocol for the design of 'better' peptides is set. The PepDesign procedure calculates new peptides fulfilling the hypothesis, tests the conformational space of these peptides in interaction with the target by angular dynamics and goes up to the selection of the best peptide based on the analysis of complex structure properties. Experimental biological assays are finally used to test the selected peptides, hence to validate the approach. Applications of PepDesign are wide because the procedure will remain similar irrespective of the target which can be a protein, a drug or a nucleic acid. In this paper, we describe the design of peptides which binds to the fusogenic helical form of the C-terminal domain of the Abeta peptide (Abeta29-42).     

Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Intein-mediated expression is an effective approach in the study of beta-defensins

Mammalian beta-defensins are an important family of host defense peptides with diverse functions. Surprisingly most of the mammalian beta-defensin genes are revealed preferentially expressed in the male organs. There is a pressing need to understand how the ample defensin repertoires work in both host defense and fertility with an aim to overcome antibiotic resistance of pathogens and reproductive problems. The biggest obstacle is the production of beta-defensin peptides as beta-defensins are small, antimicrobial and multi-disulfide molecules. In this study, the well documented HBD2, function-unknown RBD1 and function-partly-known rBin1b are successfully expressed and assayed. This approach overcomes the difficulties in beta-defensin production and provides a convenient and economical peptide-production platform to elucidate the antimicrobial activities and clinical prospects of beta-defensins. In the strategy of recombinant expression, this approach may be the best to develop the "natural" peptide pools for both host defense and fertility in a cost-effective manner.     

Phosphotyrosine profiling of human cerebrospinal fluid

Background

Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.

Methods

In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.

Results

To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.

Conclusions

The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.

     

PICarver: a software tool and strategy for peptides isoelectric focusing

The use of isoelectric focusing as first dimension of separation is a new trend in shotgun proteomics. In all applications using this approach, peptides are separated into equitable fractions, whereas theoretical distribution of peptides according to p I is heterogeneous. We present the development of a new tool and strategy that generates a fractionation scheme resulting in almost even distribution of peptides per fraction, based on theoretical and experimental data. The "pICarver" software tool also increases the throughput of the approach by reducing the number of fractions and merging the peptide-poor regions. A set of isoelectric point fluorescent peptide markers was also developed in combination with the pICarver program to calibrate the pH gradient of commercially available strips. These markers enhanced the precision of pICarver predications. The overall strategy allowed detecting false positive identification and post-translational modifications. The software tool is freely available on www.expasy.org/tools/pICarver.     

Powerful workhorses for antimicrobial peptide expression and characterization

Discovery of antimicrobial peptides (AMP) is to a large extent based on screening of fractions of natural samples in bacterial growth inhibition assays. However, the use of bacteria is not limited to screening for antimicrobial substances. In later steps, bioengineered "bugs" can be applied to both production and characterization of AMPs. Here we describe the idea to use genetically modified Escherichia coli strains for both these purposes. This approach allowed us to investigate SpStrongylocins 1 and 2 from the purple sea urchin Strongylocentrotus purpuratus only based on sequence information from a cDNA library and without previous direct isolation or chemical synthesis of these peptides. The recombinant peptides are proved active against all bacterial strains tested. An assay based on a recombinant E. coli sensor strain expressing insect luciferase, revealed that SpStrongylocins are not interfering with membrane integrity and are therefore likely to have intracellular targets.     

Improving the efficiency of evolutionary de novo peptide design: strategies for probing configuration and parameter settings

Evolutionary molecular design based on genetic algorithms (GAs) has been demonstrated to be a flexible and efficient optimization approach with potential for locating global optima. Its efficacy and efficiency are largely dependent on the operations and control parameters of the GAs. Accordingly, we have explored new operations and probed good parameter setting through simulations. The findings have been evaluated in a helical peptide design according to "Parameter setting by analogy" strategy; highly helical peptides have been successfully obtained with a population of only 16 peptides and 5 iterative cycles. The results indicate that new operations such as multi-step crossover-mutation are able to improve the explorative efficiency and to reduce the sensitivity to crossover and mutation rates (CR-MR). The efficiency of the peptide design has been furthermore improved by setting the GAs at the good CR-MR setting determined through simulation. These results suggest that probing the operations and parameter settings through simulation in combination with "Parameter setting by analogy" strategy provides an effective framework for improving the efficiency of the approach. Consequently, we conclude that this framework will be useful for contributing to practical peptide design, and gaining a better understanding of evolutionary molecular design.     

Thermolabile H-2Kb molecules expressed by transporter associated with antigen processing-deficient RMA-S cells are occupied by low-affinity peptides.

RMA-S cells do not express functional TAP, yet they express MHC class I molecules at the cell surface, especially at reduced temperatures (26 degrees C). It is generally assumed that such class I molecules are "empty," devoid of any associated peptide. A radiochemical approach was used to label class I-associated peptides and to determine the extent to which Kb molecules in RMA-S cells are associated with peptides. These studies revealed that at 26 degrees C Kb molecules in RMA-S cells are occupied with self-peptides. Such peptides stably associate with Kb at 26 degrees C but easily dissociate from them at 37 degrees C, suggesting low-affinity interactions between Kb and the associated peptides. At 26 degrees C, at least some of these Kb molecules are stably expressed in a peptide-receptive state on the cell surface, whereas at 37 degrees C they are short lived and are only transiently capable of binding and presenting exogenously supplied OVA 257-264 peptide for presentation to CD8+ Kb-restricted T lymphocytes. Thus contrary to current models of class I assembly in TAP-deficient RMA-S cells, the presumably "empty" molecules are in fact associated with peptides at 26 degrees C. Together, our data support the existence of an alternative mechanism of peptide binding and display by MHC class I molecules in TAP-deficient cells that could explain their ability to present Ag.     

Manipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening

A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates.     

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

Snapshot Peptidomics of the Regulated Secretory Pathway

Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PC-mediated processing have been described ((R/K)X_n(R/K) where n = 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine α-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.The generation of peptide hormones or neuropeptides involves the proteolytic processing of precursor proteins by specific proteases. In neurons and endocrine cells, most, if not all, of these bioactive peptides are generated within the RSP¹ in which the processing enzymes PC1/3 or PC2 cleave precursors at basic residues (1, 2). The PC-mediated cleavage most often occurs at consecutive basic residues, but not all basic residues serve as PC recognition sites (2). This is partly because the secondary structure of a precursor also affects the substrate recognition (3). Identification of processing sites is hence a prerequisite for locating unknown peptides hidden in a precursor sequence.Peptidomics has been advocated to comprehensively study peptides cleaved off from precursor proteins by endogenous proteases (4–6). These naturally occurring peptides are beyond the reach of current proteomics and should be analyzed in their native forms. Unlike proteomics, peptidomics has the potential to uncover processing sites of precursor proteins. Most peptidomics studies, which target tissue peptidomes from brain or endocrine organs (7–11), have provided limited information about secretory peptides that could help to identify processing sites; they are too often blurred by subsequent actions of exopeptidases (cutting off a single amino acid or dipeptide from either end of a peptide).In MS-based identification of bioactive peptides present in biological samples, their relative low abundance in a total pool of naturally occurring peptides should be considered. Once extracted from cultured cells or tissues, bona fide secretory peptides and nonsecretory peptides or peptide fragments caused by degradation of abundant cytosolic proteins cannot be discriminated, and therefore we need to analyze samples rich in secretory peptides to facilitate the identification of bioactive peptides. Several attempts have been made to isolate secretory proteins or peptides, such as subcellular fractionation for harvesting secretory granules (12, 13). With all these efforts, a limited number of secretory peptides have been identified, and many known bioactive peptides still escape analysis.We took advantage of the fact that peptides processed in the RSP are enriched in secretory granules of neurons and endocrine cells and released on exocytosis. Here we applied a brief exocytotic stimulus (2 min) to cultured human endocrine cells and identified peptides released into supernatant using LC-MSMS on an LTQ-Orbitrap mass spectrometer. Nearly 97% of the identified peptides arose from precursor proteins known to be recruited to the RSP, such as peptide hormone precursors and granin-like secretory proteins. Our approach was validated by the identification of previously known processing sites of peptide hormone precursors. In addition, a majority of the identified peptides retained cleavage sites that agree with consensus cleavage sites for PCs, which are informative enough to deduce the processing sites of RSP proteins. This peptidomics approach will expedite the identification of unknown bioactive peptides.     

P99-T A Simple Method to Quantitate Selenomethionine Incorporation into Proteins

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.¹ Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.     

Designer natriuretic peptides: a vision for the future of heart failure therapeutics

Despite recent pharmacological advances in heart failure therapy, mortality from acute decompensated heart failure remains high. Conventional therapies are often insufficient to address the complex interplay between structural, functional, neurohumoral, and renal mechanisms involved in the heart failure syndrome. The natriuretic peptide system, however, offers a unique pleiotropic strategy which could bridge this gap in heart failure therapy. Exogenous administration of native A-type and B-type natriuretic peptides has been met with both success and limitations, and despite the limitations, remains a worthwhile endeavor. Alternatively, synthetic modification to create "designer" chimeric peptides holds the possibility to extend both the application and therapeutic benefits possible with a natriuretic peptide based approach. Herein we describe the development of natriuretic peptide based heart failure therapies, including the design, rationale, and preliminary studies of the novel chimeric peptides CD-NP and CU-NP.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

Amyloid‐like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid‐like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid‐like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG‐active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid‐like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label‐free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.