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PubMed1999

The Aplysia mytilus inhibitory peptide-related peptides: identification, cloning, processing, distribution, and action.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

Amino Acid Sequence↗
PubMed1999

beta-lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties of hydrolysate fractions formed by the action of plasmin.

beta-Lactoglobulin (betaLg) was hydrolyzed by plasmin to a degree of hydrolysis of 4%. The hydrolysate was fractionated by ion-exchange chromatography and subsequent hydrophobic-interaction chromatography. The betaLg peptide fraction consisting of smaller peptides (mostly <2 kDa) had poor foam- and emulsion-forming and -stabilizing properties. Most of the betaLg peptides were identified (in either the nonreduced or reduced form) by mass spectrometry on the basis of the known primary structure of the intact protein and the specificity of the enzyme. The peptides formed during betaLg/plasmin-hydrolysis were (1) peptides lacking a cysteyl residue, (2) peptides composed of a single amino acid chain containing intramolecular disulfide bonds, and (3) peptides composed of two amino acid chains linked by an intermolecular disulfide bond. It appeared that significant SH/SS-exchange had taken place during hydrolysis. Many of the peptides present in the peptide fraction that exhibited good functional properties were disulfide-linked fragments.

Amino Acid Sequence↗
PubMed1996

Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity.

The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase domain was expressed in Escherichia coli, purified, denatured, and refolded to an enzymatically active form. We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid substitution within the designated peptide identified residues I29 and I25 as critical for potential cofactor activity. We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid peptide (aa 22-33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development.

Amino Acid Sequence↗
PubMed2006

A fast coarse filtering method for peptide identification by mass spectrometry.

MOTIVATION: We reformulate the problem of comparing mass-spectra by mapping spectra to a vector space model. Our search method leverages a metric space indexing algorithm to produce an initial candidate set, which can be followed by any fine ranking scheme. RESULTS: We consider three distance measures integrated into a multi-vantage point index structure. Of these, a semi-metric fuzzy-cosine distance using peptide precursor mass constraints performs the best. The index acts as a coarse, lossless filter with respect to the SEQUEST and ProFound scoring schemes, reducing the number of distance computations and returned candidates for fine filtering to about 0.5% and 0.02% of the database respectively. The fuzzy cosine distance term improves specificity over a peptide precursor mass filter, reducing the number of returned candidates by an order of magnitude. Run time measurements suggest proportional speedups in overall search times. Using an implementation of ProFound's Bayesian score as an example of a fine filter on a test set of Escherichia coli protein fragmentation spectra, the top results of our sample system are consistent with that of SEQUEST.

Algorithms↗
PubMed1988

Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP.

Amino Acid Sequence↗
PubMed2002

Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution.

Profilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28-30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis.

Animals↗
PubMed2000

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42.

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.

Amyloid beta-Peptides↗
PubMed2003

Protein and peptide identification: the rôle of mass spectrometry in proteomics.

Proteomics encompasses several disciplines in the quest to unravel the science of life. Mass spectrometry plays a key rôle in proteomics-associated analyses and this review describes that function in terms of the instrumentation used, the results generated, data interpretation and the advances in analytical techniques being made to deal with the production of highly specific, high throughput data. The review has 151 references.

Mass Spectrometry↗
PubMed2001

Clonal expansion of freshly isolated CD4T cells by randomized peptides and identification of peptide ligands using combinatorial peptide libraries.

We synthesized Xn (n = 9 -- 19) peptides that consist of 9 to 19 residues with random sequences. X19 is considered to deliver antigenic stimuli to CD4 T cells, because: (a) X19 induces proliferation of peripheral blood mononuclear cells (PBMC), in the presence of IL-2, which is abrogated by monoclonal antibodies to class II HLA; (b) X19 + IL-2 induces proliferation of CD4 T cell clones of distinct specificities; and (c) T cell clones recognizing the same TCR ligands with distinct V beta usage are equally stimulated by X19 + IL-2. We next co-cultured single peripheral CD4 T cells with X19 and mitomycin-treated autologous PBMC. Indeed, single T cells of CD45RA(-) memory phenotype exhibited clonal expansion, with variable rates of proliferation, when IL-4, IL-7, IL-9, IL-15 and agonistic antibody to CD29 were included in the culture. These T cell clones showed heterogeneous proliferation patterns against KGXXXXXXXXXGK-based and KGXXXXXXXXXGKGKK-based combinatorial peptides libraries, in the presence of IL-2. Pattern-match search on a T cell clone resulted in peptide ligand candidates, one of which induced proliferation, as did protein molecules carrying the corresponding sequence. These results indicate that X19 can induce proliferation of peripheral memory T cells, the peptide ligands of which can be determined using combinatorial peptide libraries.

Amino Acid Sequence↗
PubMed1994

Major histocompatibility complex class I allele-specific peptide libraries: identification of peptides that mimic an H-Y T cell epitope.

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.

Amino Acid Sequence↗
PubMed2006

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA.

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Amino Acid Sequence↗
PubMed2006

Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis.

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.

Chemical Fractionation↗
PubMed1998

Identification of peptides for immunotherapy of cancer. It is worth the effort.

As the nature of the T cell immune response is defined by T cell receptor recognition of small protein fragments, referred to as peptides, the identification of peptides would lead us to understanding and directing the T-cell-mediated immune response. Immunogenic peptides might be used for vaccination and activation of the immune reaction against cancer- and virus-infected cells. Additionally, the knowledge of immunogenic peptides was expected to lead to blocking of allergic reactions and autoimmune diseases. Based on these assumptions, the search for immunogenic peptides was started in mice and man in the mid-1980s. After a decade of peptide identification and testing in vitro and in vivo, this may be a proper time to evaluate the results from the peptide-related work and determine the possible applications of this knowledge for the next decade. In this review we discuss the identification of peptides, their use in murine models, as well as clinical data from peptide vaccinations or therapies. Potential hazards and limitations of peptide use in immunotherapy and other possible applications for peptides or peptide motifs in immunotherapy are evaluated.

Animals↗
PubMed2002

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Antigens, Neoplasm↗
PubMed2003

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.

Algorithms↗