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PubMed2005

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules.

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.

Affinity Labels↗
PubMed2002

V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein.

V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the alpha and beta subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1.CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of approximately 1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.

Actin Depolymerizing Factors↗
PubMed2004

Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography.

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

Animals↗
PubMed2006

[Identification of serum biomarkers for ovarian cancer using protein chips and time of flight mass spectrometry technology].

OBJECTIVE: To screen relatively specific biomarkers in serum of ovarian cancer patients using surface-enhanced laser desorption and ionization time of flight mass spectrometry and protein chip technology. METHODS: Serum samples from 99 ovarian cancer patients, 87 healthy volunteers and 21 patients with other ovarian diseases were analyzed using metal affinity chromatography protein chip IMAC3 and cation exchange protein chip WCX2, which can specifically bind the metal-combining-proteins. Proteomic spectra were generated by mass spectrometry. The peaks were detected and filtrated by Ciphergen proteinchip software 3.2.0. Using the Biomarker Pattern 5.0 software, a diagnostic system was developed for separating ovarian cancer from the healthy group. RESULTS: Thirty-one protein peaks were significantly different between ovarian cancer patients and healthy controls (P < 0.05). A diagnostic model consisting of protein peaks from IMAC3 and WCX2 was established. Large scale blind test generated a sensitivity of 100% and specificity of 98% respectively. A differentially expressed protein with a mass-to-charge rate of 7769 was identified as potential biomarkers to distinguish ovarian cancer from other ovarian diseases. CONCLUSIONS: Time of flight mass spectrometry is a useful tool for the detection and identification of new protein markers in serum. It will provide a highly accurate and innovative approach for the diagnosis of ovarian cancer.

Adult↗
PubMed2006

Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress.

Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species (ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA. But aggregation and cross-linking can occur as well, leading to a series of problems including disruption of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation occurs and the macromolecular species involved are poorly understood. In the study reported here, evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since they were not in direct contact, it was not clear how this could occur unless it was through the RNA separating them in the ribosome. This was confirmed in a multiple-step process, the first being derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured were then selected again using boronate affinity chromatography to capture vicinal diol-containing proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue with 2',3'-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA. Aggregation of proteins with ribosomal RNA has not been previously reported. The probable impact of this phenomenon cells is to diminish the protein synthesis capacity.

Biotin↗
PubMed2005

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites.

BACKGROUND: Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. RESULTS: Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. CONCLUSION: A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

Journal Article↗
PubMed2006

Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae.

Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.

Affinity Labels↗
PubMed2004

A proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality.

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

Binding Sites↗
PubMed2005

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach.

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.

Endoribonucleases↗
PubMed2005

Phosphoproteomics in analyzing signaling pathways.

A surprisingly fewer than expected number of genes in the human genome suggests that sophistication of its biologic system is, in part, due to complex regulation of protein activities. The activities of most cellular proteins are regulated by post-translational modifications. One of the most important post-translational modifications is reversible protein phosphorylation, which decorates more than 30% of the proteome and regulates signal transduction pathways under normal conditions as well as in disorders such as diabetes, neurodegenerative diseases, autoimmune diseases and several forms of cancers. This review examines the recent developments in mass spectrometry-based methods for phosphoproteome analysis and its applications for the study of signal transduction pathways. The basic principles of non-mass spectrometry-based methods, such as chemical genetics and flow cytometry-based approaches, are also discussed as well as their specific advantages to signaling studies. Finally, signaling pathways are discussed in the light of large-scale protein interaction studies. The proteomic methods addressed in this review are emerging as some of the essential components in systems biology, which seeks to describe signaling networks through integration of diverse types of data and, in the future, to allow computational simulations of complex biologic pathways in health and disease.

Chromatography, Affinity↗
PubMed2002

Comprehensive proteomic analysis of the human spliceosome.

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

Amino Acid Motifs↗
PubMed2006

Proteomic characterization of human serum proteins associated with the fat-derived hormone adiponectin.

Adiponectin is a fat cell-secreted hormone with antidiabetic and anti-inflammatory activities. The reduced adiponectin levels are associated with obesity-related metabolic syndrome. Replenishment of this hormone into animal models can improve insulin sensitivity, decrease blood glucose and lipid levels, and prevent the development of atherosclerosis and fatty liver injury. Despite these findings, the underlying molecular mechanisms remain largely unknown. Here, we have used affinity chromatography to purify the protein complexes that are associated with adiponectin in human serum. The nature of these adiponectin-binding proteins was analyzed by MS/MS. Eight proteins from the adiponectin-containing protein mixtures have been identified. Many of them, including thrombospondin-1 (TSP-1), histidine-rich glycoprotein, kininogen 1, and alpha 2 macroglobulin (alpha2M), are well-known glycoproteins involved in the regulation of inflammation, angiogenesis, and tissue remodeling. Coimmunoprecipitation and radioligand competitive-binding assays confirmed the direct interactions between adiponectin and alpha2M, or TSP-1. Moreover, these specific bindings were also detected in the serum samples derived from both healthy human subjects and patients with type 2 diabetes. In summary, our study demonstrated that, in the circulation, adiponectin forms protein complexes with other serum proteins. These proteins might serve as the physiological-binding partners of adiponectin and regulate its bioavailability and biological activities.

Adiponectin↗
PubMedDate not supplied

Capture of peptides with N-terminal serine and threonine: a sequence-specific chemical method for Peptide mixture simplification.

The objective of this study was to evaluate a sequence-specific chemistry for the ability to specifically capture peptides that contain N-terminal serine or threonine residues from mixtures. The first step is the oxidation of the 1,2-amino alcohol structure -CH(NH(2))CH(OH)- of peptides containing N-terminal serine or threonine with periodate. The newly formed aldehyde reacts with a labeling reagent containing a hydrazide, RCONHNH(2), to form a hydrazone-peptide conjugate, RCONHN=CH-peptide. Biotin-labeled conjugates can then be isolated by affinity purification with streptavidin. The method described in this report can be useful in simplifying the complex mixtures of peptides that are generated in typical proteomic analysis, where proteins are digested with trypsin and analyzed using liquid chromatography mass spectrometry data. The sequence-specific peptide selection not only reduces the complexity of digest mixtures, but also provides additional information for peptide identification. The targeted peptides are those that have either serine or threonine adjacent to a protease cleavage site. The sequence information should greatly aid in both database matching for protein identification and for de novo sequence determination.

Affinity Labels↗
PubMed2004

Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt(+2) immobilized metal affinity chromatography.

Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.

Amino Acid Sequence↗
PubMed2006

Neuroproteomics: relevance to anxiety disorders.

Despite advances in the treatment of anxiety disorders, there is a need for medications with greater efficacy and fewer side effects. Advances in techniques to facilitate high throughput, mass analysis of proteins potentially allows for new drug targets, with a shift in focus from membrane receptor proteins and enzymes of neurotransmitter metabolism to molecules in intracellular signal transduction and other pathways. A computerized literature search was done to collect studies on recently developed proteomic techniques and their application in psychiatric research. Particular techniques, such as two-dimensional electrophoresis, two-dimensional differential gel electrophoresis, isotope-coded affinity tags, and isotope tags for relative and absolute quantification, are reviewed. In addition, a combination of these techniques with MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analysis is discussed in relation to possible novel signaling pathways relevant to anxiety disorders, and to the development of biomarkers for the evaluation of these conditions.

Animals↗
PubMed2005

Unraveling kinase signaling pathways with chemical genetic and chemical proteomic approaches.

Protein kinases are involved in a wide variety of physiological and pathological processes. Small kinase inhibitor molecules are frequently used to dissect signaling pathways or to counteract oncogenic events. However, in many cases the precise role of a given inhibitor is not well understood. Besides a known primary target, potential secondary targets might be involved in the biological response to the drug. We describe recent advances in chemical genetics and chemical proteomics that allow a new comprehensive analysis of kinase signaling pathways. One approach consists of engineering the kinase pocket to design inhibitor sensitive and resistant alleles. A second strategy is to modify the kinase pocket to specifically accept radio-labeled ATP analogs, allowing the identification of direct downstream substrates after transphosphorylation. The third method takes advantage of recently improved inhibitor affinity chromatography to identify inhibitor targets by mass spectrometry. Ultimately, these new technologies will help define the actions of kinase inhibitors useful for human disease treatment.

Adenosine Triphosphate↗
PubMed2003

Fractionation of cytosolic proteins on an immobilized heparin column.

Currently there is great interest in the development of methods to simplify complex protein mixtures for analysis by proteomic strategies. The objective of this study was to develop and evaluate immobilized heparin chromatography to simplify such mixtures and to enrich minor proteins. The method is evaluated with cytosol from human breast cancer MCF-7 cells. This protein mixture was fractionated into three portions and eluted with a stepwise salt gradient. These were characterized by protein analysis, two-dimensional gel electrophoresis, and mass spectrometry, with attention to reproducibility, overlap between fractions, simplification of protein mixtures, and enrichment of low-abundance proteins. It was possible to identify proteins enriched in the fractionated mixtures that were not even detectable in gel arrays of the total cytosol. The method was shown to be suitable for integration with other proteomics strategies.

Animals↗