共查询到20条相似文献,搜索用时 31 毫秒
1.
A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure under dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-of-use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an alpha-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as 'protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These studies imply that the dry peptide array system is a promising tool for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies. 相似文献
2.
Kracun SK Cló E Clausen H Levery SB Jensen KJ Blixt O 《Journal of proteome research》2010,9(12):6705-6714
Identification of disease-specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I(2)/NaBH(4) and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MS(n). As proof-of-principle, tumor -specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor-specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were resynthesized at the preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid discovery of O-glycopeptide biomarkers and the method has applicability in other types of assays such as lectin/antibody/enzyme specificity studies as well as investigation of other PTMs. 相似文献
3.
Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase possibilities of their design and improve their preparation and separation. Sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). Part I deals with the development of various structural forms of MAPs as well as their application as antigens, immunogens, and for immunodiagnostic and biochemical purposes. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
4.
Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
5.
Large-scale functional analysis using peptide or protein arrays 总被引:22,自引:0,他引:22
The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens. Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells. Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities. In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions. Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence. 相似文献
6.
The complementary fragments of human Hb α, α1–30, and α31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide
splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated
by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction.
The segments α24–30 and α31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous
segment (product) α24–40 has been manipulated by engineering the amino acid replacements to the positions α27 and α31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment α24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies
have confirmed that the side chains at α27 and α31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends
on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute
up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic
stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino
acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the
presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability
to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions
of peptides could be designed into products to facilitate the modular assembly of peptides/proteins. 相似文献
7.
The complementary fragments of human Hb α, α1–30, and α31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide
splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated
by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction.
The segments α24–30 and α31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous
segment (product) α24–40 has been manipulated by engineering the amino acid replacements to the positions α27 and α31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment α24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies
have confirmed that the side chains at α27 and α31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends
on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute
up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic
stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino
acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the
presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability
to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions
of peptides could be designed into products to facilitate the modular assembly of peptides/proteins. 相似文献
8.
Robin Hurst 《Analytical biochemistry》2009,392(1):45-53
Protein arrays hold great promise for proteome-scale analysis of protein-protein interaction networks, but the technical challenges have hindered their adoption by proteomics researchers. The crucial issue of design and fabrication of protein arrays have been addressed in several studies, but the detection strategies used for identifying protein-protein interactions have received little attention. In this study, we evaluated six different detection strategies to identify four different protein-protein interaction pairs. We discuss each detection approach in terms of signal-to-background (S/B) ratio, ease of use, and adaptability to high-throughput format. Protein arrays for this study were made by expressing both the bait proteins (proteins captured at the surface) and prey proteins (probes) in cell-free rabbit reticulocyte lysate (RRL) systems. Bait proteins were expressed as HaloTag fusions that allow covalent capture on a HaloTag ligand-coated glass without any prior protein purification step. Prey proteins were expressed and modified with either tags (protein or peptides) or labels (fluorescent or radiometric) for detection. This simple method for creating protein arrays in combination with our analyses of several detection strategies should increase the usefulness of protein array technologies. 相似文献
9.
In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in
the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms,
progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein
analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative
detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the
need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing
proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate
the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes
the basic technologies that are needed in the fabrication of protein arrays and their recent applications. 相似文献
10.
To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips. 相似文献
11.
Background
Members of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins. 相似文献12.
Protein domain frequency and distribution among kingdoms was statistically analyzed using the SCOP structural database. It
appeared that among chosen protein domains with the best resolution, eukaryotic proteins more often belong to α-helical and
β-structural proteins, while proteins of bacterial origin belong to α/β structural class. Statistical analysis of folding
rates of 73 proteins with known experimental data revealed that bacterial proteins with simple kinetics (23 proteins) exhibit
a higher folding rate compared to eukaryotic proteins with simple folding kinetics (27 proteins). Analysis of protein domain
amino acid composition showed that the frequency of amino acid residues in proteins of eukaryotic and bacterial origin is
different for proteins with simple and complex folding kinetics. 相似文献
13.
Daisuke Fujiwara Zhengmao Ye Masaki Gouda Koichi Yokota Takeshi Tsumuraya Ikuo Fujii 《Bioorganic & medicinal chemistry letters》2010,20(5):1776-1778
Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix–loop–helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family. 相似文献
14.
Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, protein-nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening. 相似文献
15.
Han Hu Kshitij Khatri Joshua Klein Nancy Leymarie Joseph Zaia 《Glycoconjugate journal》2016,33(3):285-296
Despite the publication of several software tools for analysis of glycopeptide tandem mass spectra, there remains a lack of consensus regarding the most effective and appropriate methods. In part, this reflects problems with applying standard methods for proteomics database searching and false discovery rate calculation. While the analysis of small post-translational modifications (PTMs) may be regarded as an extension of proteomics database searching, glycosylation requires specialized approaches. This is because glycans are large and heterogeneous by nature, causing glycopeptides to exist as multiple glycosylated variants. Thus, the mass of the peptide cannot be calculated directly from that of the intact glycopeptide. In addition, the chemical nature of the glycan strongly influences product ion patterns observed for glycopeptides. As a result, glycopeptidomics requires specialized bioinformatics methods. We summarize the recent progress towards a consensus for effective glycopeptide tandem mass spectrometric analysis. 相似文献
16.
Wingren C Steinhauer C Ingvarsson J Persson E Larsson K Borrebaeck CA 《Proteomics》2005,5(5):1281-1291
Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics. 相似文献
17.
Christine Talmard Rodrigue Leuma Yona Peter Faller 《Journal of biological inorganic chemistry》2009,14(3):449-455
The amyloidoses are a group of disorders characterized by aberrant protein folding and assembly, leading to the deposition
of insoluble protein fibrils (amyloid), which provokes cell dysfunction and later cell death. One of the physiologically relevant
environmental factors able to affect the conformation and hence the aggregation properties of amyloidogenic proteins/peptides
is metal ions. Zn(II) promotes aggregation of most amyloidogenic peptides/proteins in vitro, including amyloid β protein (Aβ),
but the underlying mechanism is not known. To better understand this mechanism the present study focused on the partially
α-helical conformer, supposed to be an intermediate in Aβ aggregation. This partially α-helical conformer is stabilized by
10–20% 2,2,2-trifluoroethanol (TFE): therefore, the influence of Zn binding on the aggregation of the amylidogenic model peptide
Aβ(1–28) (Aβ28) was investigated at different TFE concentrations. The results showed a synergistic effect of Zn(II) and 10%
TFE, i.e., that either Zn or 10% TFE accelerated Aβ28 aggregation on its own, but with them together an at least 10 times
promotion of Aβ28 aggregation was observed. Further studies by thioflavin T fluorescence spectroscopy, transmission electron
microscopy, and circular dichroism (CD) spectroscopy suggested that the aggregates of Zn-Aβ28 formed in 10%TFE contain a β-sheet
secondary structure and are more of the amyloid type. CD spectroscopy indicated that Zn binding disrupted partially the α-helical
structure of Aβ28 in TFE. Thus, we propose that the promotion of Aβ28 aggregation by Zn is based on the transformation of
the partially α-helical conformer (intermediate) towards the β-sheet amyloid structure by a destabilization of the α-helix
in the intermediate.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
相似文献
Peter FallerEmail: Email: |
18.
C. Nadal 《Cell proliferation》1987,20(3):331-341
Abstract. The activity of a glycopeptide prepared from rat serum by treatment with trypsin and ultrafiltration was investigated in several in vivo proliferation systems. In baby rat hepatocytes synchronized by a subcutaneous injection of casein solution it caused a G1 -S block, stopping cells at the end of the G1 phase and sending them back to the G0 phase. The glycopeptide also caused a G1 -S block in young adult rats during the first semi-synchronized wave of proliferation that followed partial hepatectomy. Inhibition of hepatocyte proliferation by the glycopeptide was suppressed by blood proteins from normal rats but not from acute phase rats. α1 -acid glycoprotein, an acute phase protein, increased this inhibition and reversed the antagonistic effect of normal blood proteins. In normal baby rats a G1 -S block of non-synchronously proliferating hepatocytes was produced in two situations in which the antagonistic effect of normal blood proteins was eliminated: after treatment of the glycopeptide with leucine-aminopeptidase, and after mixing it with α1 -acid glycoprotein. The glycopeptide did not inhibit cell proliferation in kidney, submaxillary gland, or tongue epithelium. It seems to be the active component of a system that inhibits the proliferation of hepatocytes, probably by reducing their sensitivity to various mitogenic stimuli. 相似文献
19.
Michelle A. Camerino David C. M. Kong David K. Chalmers Philip E. Thompson 《International journal of peptide research and therapeutics》2008,14(4):323-331
The α-helix is one of the most common secondary structure elements adopted by proteins and is commonly stabilized in synthetic
peptides via the formation of a covalent side-chain to side-chain lactam bridge. In this study, we explored the application
of various side-chain to side-chain lactam bridges to helix stabilization of kisspeptin analogues, an interesting candidate
for ligand-based drug discovery with potential as anti-metastatic agents. We successfully synthesised a series of Asp/Lys,
Lys/Asp, Glu/Lys and Lys/Glu lactams, finding peptide (1) cyclo(4,8)Tyr-Asn-Trp-Glu-Ala-Phe-Gly-Lys-Arg-Phe-NH2, to exhibit characteristic α-helical activity in aqueous buffer, in comparison to the linear native peptide, which showed
no helical character. 相似文献
20.
Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region 总被引:6,自引:0,他引:6
Fourier transform spectroscopy in the mid-infrared (400–5,000 cm−1) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular
architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount
of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated
sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm−1) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation
and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR
spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are
discussed. 相似文献