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PubMed2006

Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond.

Yeast-based functional genomics and proteomics technologies developed over the past decade have contributed greatly to our understanding of bacterial, yeast, fly, worm, and human gene functions. In this review, we highlight some of these yeast-based functional genomic and proteomic technologies that are advancing the utility of yeast as a model organism in molecular biology and speculate on their future uses. Such technologies include use of the yeast deletion strain collection, large-scale determination of protein localization in vivo, synthetic genetic array analysis, variations of the yeast two-hybrid system, protein microarrays, and tandem affinity purification (TAP)-tagging approaches. The integration of these advances with established technologies is invaluable in the drive toward a comprehensive understanding of protein structure and function in the cellular milieu.

Forecasting↗
PubMed2006

Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors.

Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.

HeLa Cells↗
PubMed2003

Prefractionation techniques in proteome analysis.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Chemical Fractionation↗
PubMed2005

Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography.

Phosphoproteome studies are hampered by the lack of methods which allow a comprehensive and fast analysis of intact phosphoproteins. Here we describe an immobilized metal-affinity chromatography (IMAC)-based technique for the enrichment of phosphorylated proteins, which allows recovery of up to 90% of phosphoproteins. This technique is compatible with 2-DE and can be applied to cultured cells and tissues.

Cell Line↗
PubMedDate not supplied

Phosphoproteome analysis.

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

Acrylic Resins↗
PubMed2006

Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

Cell Line, Tumor↗
PubMed2005

Biochemical characterization of the native Kv2.1 potassium channel.

Functional diversity of potassium channels in both prokaryotic and eukaryotic cells suggests multiple levels of regulation. Posttranslational regulation includes differential subunit assembly of homologous pore-forming subunits. In addition, a variety of modulatory subunits may interact with the pore complex either statically or dynamically. Kv2.1 is a delayed rectifier potassium channel isolated by expression cloning. The native polypeptide has not been purified, hence composition of the Kv2.1 channel complexes was not well understood. Here we report a biochemical characterization of Kv2.1 channel complexes from both recombinant cell lines and native rat brain. The channel complexes behave as large macromolecular complexes with an apparent oligomeric size of 650 kDa as judged by gel filtration chromatography. The molecular complexes have distinct biochemical populations detectable by a panel of antibodies. This is indicative of functional heterogeneity. Despite mRNA distribution in a variety of tissues, the native Kv2.1 polypeptides are more abundantly found in brain and have predominantly Kv2.1 subunits but not homologous Kv2.2 subunits. The proteins precipitated by anti-Kv2.1 and their physiological relevance are of interest for further investigation.

Amino Acid Sequence↗
PubMed2006

Ribosome display for improved biotherapeutic molecules.

Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein-protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.

Animals↗
PubMed2003

Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics.

During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.

Animals↗
PubMed2003

Stable isotope-coded proteomic mass spectrometry.

Developing the ability to quantify changes in protein abundance between cells subjected to a variety of physiological and environmental conditions is an extremely active area of proteome research. Although advances in chromatography, mass spectrometry instrumentation, and bioinformatics have contributed to producing a viable method for comparative proteome-wide analyses, the highest precision of quantitation is based, in part, upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications using stable isotope labeling has been achieved, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.

Affinity Labels↗
PubMed2006

The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay.

Proteins that interact with nucleic acids are central to numerous cellular processes, and their continuing characterization represents one of the foremost challenges in the postgenomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.

Amino Acid Sequence↗
PubMed2002

Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Affinity Labels↗
PubMed2003

Proteomic analysis of proteins that binds specifically to the homologous repeat regions of white spot syndrome virus.

White spot syndrome virus (WSSV) is a major pathogen in the cultivated shrimp. Nine homologous repeat regions (hrrs) have been reported interspersed throughout the WSSV genome. In this investigation, the protein fraction that specifically bound to the hrrs was isolated by using DNA-affinity chromatography. A total of 9 (S1 to S9) and 5 (C1 to C5) proteins separated from the WSSV infected shrimp and the healthy shrimp, respectively, were detected by using two-dimensional polyacrylamide gel electrophoresis, and 6 proteins changed with WSSV infection were analyzed by mass spectrometry (MS). One (S4) of the 6 proteins examined was identified as WSSV ORF59 protein, and another (S3) was a shrimp arginine kinase. No homologous proteins were found with the remaining 4 proteins by searching in the WSSV ORF database and NCBI database. The specific binding site of the 6 proteins was then determined by gel mobility shift assay (GMSA). Temporal analysis revealed that ORF59 gene was transcribed at the early stage of the infection. The results we obtained provide important information to understand WSSV replication. The combination of DNA-affinity chromatography, 2D-PAGE and MS approaches should have general application to the identification of gene regulating proteins of WSSV. The results represent the first isolation of a set of proteins that bind to the hrrs, and, furthermore, lead us a new research direction for the prevention and the therapy of WSSV.

Animals↗
PubMed2006

Trypanosoma seryl-tRNA synthetase is a metazoan-like enzyme with high affinity for tRNASec.

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma. First, the biological function of a putative Trypanosoma seryl-tRNA synthetase was characterized in vivo. Secondly, the molecular recognition by Trypanosoma seryl-tRNA synthetase of its cognate tRNAs was dissected in vitro. The cellular distribution of tRNA(Sec) was studied, and the catalytic constants of its aminoacylation were determined. These were found to be markedly different from those reported in other organisms, indicating that this reaction is particularly efficient in trypanosomatids. Our functional data were analyzed in the context of a new phylogenetic analysis of eukaryotic seryl-tRNA synthetases that includes Trypanosoma and Leishmania sequences. Our results show that trypanosomatid seryl-tRNA synthetases are functionally and evolutionarily more closely related to their metazoan homologous enzymes than to other eukaryotic enzymes. This conclusion is supported by sequence synapomorphies that clearly connect metazoan and trypanosomatid seryl-tRNA synthetases.

Acylation↗
PubMed2006

Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption.

A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.

Adsorption↗
PubMed2005

Multidimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phase of the human serum proteome: optimization and protein database.

In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic "cellular" proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present.

Albumins↗
PubMedDate not supplied

Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins.

Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10(-12) M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC-MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI-MS or LC-MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 microg of protein well tolerated using a 4.6 mm ID x 50 mm column, or up to 1.5 mg on a 9.4 mm ID x 50 mm column. Elevated column temperature (80 degrees C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC-MS methods.

Amino Acid Sequence↗
PubMed2004

Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.

To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Epidermal Growth Factor↗