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At least 721 records · Page 40Linked to original sources
PubMed2004

Identification of monoclonal anti-HMW-MAA antibody linear peptide epitope by proteomic database mining.

An efficient strategy is presented for the identification of antigenic sequences in the context of given MHC molecules of interest. The proteomic analysis of the antigenic peptide repertoire is described and demonstrated by using high-molecular weight melanoma-associated antigen. The identification of the epitopic sequence of a monoclonal antibody raised against the 250 kDa tumor associated antigen was reached by using only seven short synthetic peptide fragments, instead of the 155 non-overlapping 15-mer peptides theoretically necessary as minimum screening library. The present result has been obtained by applying as driving criteria the analysis of the peptide affinity to MHC class II molecules and the non-self discrimination concept.

Amino Acid Sequence↗
PubMed2002

Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification.

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).

Chromatography, Affinity↗
PubMed2002

Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method.

Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method. Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation.

Alkylation↗
PubMed2004

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

14-3-3 Proteins↗
PubMed2006

Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin.

Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.

Arabidopsis↗
PubMed2005

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.

Amino Acid Sequence↗
PubMed2005

Decreased levels of CXC-chemokines in serum of benzene-exposed workers identified by array-based proteomics.

Benzene is an important industrial chemical and environmental contaminant that causes leukemia. To obtain mechanistic insight into benzene's mechanism of action, we examined the impact of benzene on the human serum proteome in a study of exposed healthy shoe-factory workers and unexposed controls. Two sequential studies were performed, each using sera from 10 workers exposed to benzene (overall mean benzene air level >30 ppm) and 10 controls. Serum samples were subjected to anion-exchange fractionation and bound to three types of ProteinChip arrays (Ciphergen Biosystems, Fremont, CA) [hydrophobic (H50), metal affinity (IMAC3-Cu), and cation exchange (WCX2)]. Protein-expression patterns were detected by surface-enhanced laser desorption/ionization (SELDI)-TOF MS. Three proteins (4.1, 7.7, and 9.3 kDa) were consistently down-regulated in exposed compared with control subjects in both studies. All proteins were highly inversely correlated with individual estimates of benzene exposure (r > 0.75). The 7.7- and 9.3-kDa proteins were subsequently identified as platelet factor (PF)4 and connective tissue activating peptide (CTAP)-III. Initial proteomic results for PF4 and CTAP-III were subsequently confirmed in a single experiment using a ProteinChip-array-based immunoassay(Ciphergen Biosystems). The altered expression of the platelet-derived CXC-chemokines (40% and 63% for PF4 and CTAP-III, respectively) could not be explained by changes in absolute platelet counts. Thus, SELDI-TOF analysis of a limited number of exposed and unexposed subjects revealed that lowered expression of PF4 and CTAP-III proteins is a potential biomarker of benzene's early biologic effects and may play a role in the immunosuppressive effects of benzene.

Benzene↗
PubMed2006

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry serum protein profiling to identify nasopharyngeal carcinoma.

BACKGROUND: Diagnosis of nasopharyngeal carcinoma (NPC) at an early disease stage is important for successful treatment and improving the outcome of patients. The use of serum protein profiles and a classification tree algorithm were explored to distinguish NPC from noncancer. METHODS: Serum samples were applied to metal affinity protein chips to generate mass spectra by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Protein peak identification and clustering were performed using the Biomarker Wizard software. Proteomic spectra of serum samples from 50 NPC patients and 54 noncancer controls were used as a training set and a classification tree with 6 distinct protein masses was generated by using Biomarker Pattern software. The validity of the classification tree was then challenged with a blind test set including another 20 NPC patients and 25 noncancer controls. RESULTS: The software identified an average of 93 mass peaks/spectrum and 6 of the identified peaks were used to construct the classification tree. The classification tree correctly determined 83% (123 of 149) of the test samples with 83% (58 of 70) of the NPC samples and 82% (65 of 79) of the noncancer samples. In a combination of the serum protein profiles with Epstein-Barr (EBV) nuclear antigen 1 (EBNA1 IgA) test, the diagnostic sensitivity and specificity were increased to 99% and 96%, respectively. CONCLUSIONS: The results suggest that SELDI-TOF-MS serum protein profiles could discriminate NPC from noncancer. The combination of serum protein profiles with an EBV antibody serology test could further improve the accuracy of NPC screening.

Adult↗
PubMed2003

Profiling of the cell surface proteome.

The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations.

ADP-ribosyl Cyclase↗
PubMed2004

Target identification strategies in chemical genetics.

Chemical inhibitors have had a profound impact on many diverse fields of biology. The goal of chemical genetics is to use small molecules to perturb biological systems in a manner conceptually similar to traditional genetics. Key to the advancement of the chemical genetic paradigm is the further development of tools and approaches for the identification of the protein targets of active compounds identified in chemical genetic screens. This review will address historic examples in which forward chemical genetics yielded new insight into a biological problem through successful identification of the target of an active molecule. The approaches covered have been grouped into two broad classes: target identification by affinity-based methods and target identification by deduction. Strengths and shortcomings of each approach as it pertains to their application to modern chemical genetics will be discussed. Finally, a series of new genomic and proteomic-based techniques for target identification will be described. Although a truly general approach to target identification has yet to be developed, these examples illustrate that there are many effective strategies for successfully elucidating the biological targets of active small molecules.

DNA, Complementary↗
PubMed2005

Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen.

Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglobulin E (IgE) immunoblots. However, only a few allergens have been identified and characterized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE-reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI-TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identifying six novel allergens.

Blotting, Western↗
PubMed2006

Proteomic analysis in cancer research: potential application in clinical use.

The ultimate goal of cancer proteomics is to adapt proteomic technologies for routine use in clinical laboratories for the purpose of diagnostic and prognostic classification of disease states, as well as in evaluating drug toxicity and efficacy. The novel technologies allows researchers to facilitate the comprehensive analyses of genomes, transcriptomes, and proteomes in health and disease. The information that is expected from such technologies may soon exert a dramatic change in cancer research and impact dramatically on the care of cancer patients. Analysis of tumor-specific proteomic profiles may also allow better understanding of tumor development and the identification of novel targets for cancer therapy. The localization of gene products, which is often difficult to deduce from the sequence, can be determined experimentally. Mechanisms, such as regulation of protein function by proteolysis, recycling, and isolation in cell compartments, affect gene products, not genes. Finally, protein-protein interactions and the molecular composition of cellular structures can be determined only at the protein level. The biological variability among patient samples as well as the great dynamic range of biomarker concentrations are currently the main challenges facing efforts to deduce diagnostic patterns that are unique to specific disease states. While several strategies exist to address this problem, we have tried to offer a wide perspective about the current possibilities.

Antibodies, Neoplasm↗
PubMed2003

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation.

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.

Bacterial Outer Membrane Proteins↗
PubMed2005

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions.

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.

Acetonitriles↗
PubMed2004

Discovering protein-protein interactions.

The ongoing genomics and proteomics efforts have helped identify many new genes and proteins in living organisms. However, simply knowing the existence of genes and proteins does not tell us much about the biological processes in which they participate. Many major biological processes are controlled by protein interaction networks. A comprehensive description of protein-protein interactions is therefore necessary to understand the genetic program of life. In this tutorial, we provide an overview of the various current high-throughput methods for discovering protein-protein interactions, covering both the conventional experimental methods and new computational approaches.

Artificial Gene Fusion↗
PubMed2004

Current chemical tagging strategies for proteome analysis by mass spectrometry.

Proteomics, the analysis of the protein complement of a cell or an organism, has grown rapidly as a subdiscipline of the life sciences. Mass spectrometry (MS) is one of the central detection techniques in proteome analysis, yet it has to rely on prior sample preparation steps that reduce the enormous complexity of the protein mixtures obtained from biological systems. For that reason, a number of so-called tagging (or labeling) strategies have been developed that target specific amino acid residues or post-translational modifications, enabling the enrichment of subfractions via affinity clean-up, resulting in the identification of an ever increasing number of proteins. In addition, the attachment of stable-isotope-labeled tags now allows the relative quantitation of protein levels of two samples, e.g. those representing different cell states, which is of great significance for drug discovery and molecular biology. Finally, tagging schemes also serve to facilitate interpretation of MS/MS spectra, therefore assisting in de novo elucidation of protein sequences and automated database searching. This review summarizes the different application fields for tagging strategies for today's MS-based proteome analysis. Advantages and drawbacks of the numerous strategies that have appeared in the literature in the last years are highlighted, and an outlook on emerging tagging techniques is given.

Amino Acids↗
PubMed2006

Anion-exchange chromatography using short monolithic columns as a complementary technique for human serum albumin depletion prior to human plasma proteome analysis.

In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immunoglobulin G represent more than 75% of all such proteins. In this paper, the characterization of short monolithic columns was performed followed by the optimization of a multidimensional approach, known as conjoint liquid chromatography, to deplete human serum albumin and immunoglobulin G from a human plasma sample. Two different chromatographic modes were used: ion-exchange chromatography and affinity chromatography. A monolithic stationary phase (convective interaction media disk) bearing strong anion-exchange groups and another immobilized with protein G were placed in series into one housing. The optimal binding conditions were found that removed a majority of human serum albumin and immunoglobulin G from the human plasma sample. This method was compared to the depletion using a combination of pseudo-affinity and affinity columns. The results of the human serum albumin and immunoglobulin G depletion were confirmed by 2D electrophoresis. It has been shown that anion-exchange and affinity chromatography using convective interaction media monolithic columns can represent an efficient complementary technique for human serum albumin and immunoglobulin G removal from human plasma.

Biomarkers↗
PubMed2002

Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.

Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

Amino Acid Sequence↗