Proteomic analysis of RNA-binding proteins in dry seeds of rice after fractionation by ssDNA affinity column chromatography

In proteomic analysis, one of the major limitations is the detection of low-abundance proteins. To detect low-abundance RNA-binding proteins in mature dry seeds of rice, fractionation by single stranded DNA (ssDNA) affinity column chromatography was carried out before analysis by two-dimensional gel electrophoresis (2-DE). Proteomic analysis of the ssDNA-binding fraction revealed the existence of three types of RNA-binding proteins, including a K homology (KH) domain containing protein, a putative RNA-binding protein and a glycine-rich RNA-binding protein, in mature seeds. In addition, decreases in the putative RNA-binding protein and glycine-rich RNA-binding protein after absorbing water in seeds appear to be associated with seed germination.     

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834–840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746–2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.     

Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.     

Proteomic analysis of mammalian primary cilia

The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium-shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia-specific proteins. These possible primary cilia-specific proteins include EVC2, INPP5E, and inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.     

Proteomic identification of plant proteins probed by mammalian nitric oxide synthase antibodies

Several studies suggest that a mammalian-like nitric oxide synthase (NOS) exists in plants. Researchers have attempted to verify its presence using two approaches: (i) determination of NOS functional activity and (ii) probing with mammalian NOS antibodies. However, up to now, neither a NOS-like gene nor a protein has been found in plants. While there is still some controversy over whether the NOS functional activity seen is due to nitrate reductase, using the mammalian NOS antibodies in western blot analysis, several groups have reported the presence of immunoreactive protein bands in plant homogenates. Based on these results, immunohistochemical studies using these antibodies have also been used to localize NOS in plant tissues. However, plant NOS has never been positively identified or characterized. Thus, we used a proteomic approach to verify the identities of plant proteins that cross-reacted with the mammalian NOS antibodies. Proteins extracted from maize (Zea mays L.) embryonic axes were separated by two-dimensional gel electrophoresis and subjected to western blot analysis with the mammalian neuronal NOS and inducible NOS antibodies. Twenty immunoreactive protein spots recognized on a corresponding Coomassie blue-stained two-dimensional gel were subjected to tryptic digestion, followed by identification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Fifteen proteins were successfully identified and they have described functions that are unrelated to NO metabolism. The remaining five proteins could not be identified. The amino acid sequences of these identified proteins and those used to raise the antibodies were aligned. However, no homologous region could be found. Our results demonstrate that the mammalian NOS antibodies recognize many NOS-unrelated plant proteins. Therefore, it is inappropriate to infer the presence of plant NOS using this immunological technique.     

Proteomic analysis of phosphorylated proteins

Reversible protein phosphorylation is of crucial importance in regulatory mechanisms and signaling pathways. Novel and efficient tools and strategies are being actively developed to allow, beyond the primary identification of phosphorylated proteins, the identification of phosphorylation sites and ultimately their quantification. These approaches are being used at various scales, from studies that have dedicated and functional goals to work with more exploratory and cataloguing objectives. The information thus generated now makes it possible to use bioinformatics to revisit previous knowledge about protein phosphorylation in plants and the pertinence of available prediction models. Although the analysis of phosphorylated proteins remains a challenging task, recent success and current developments are likely to mark the transition towards the introduction of phosphoproteomics as one of the main integration levels in post-genome plant biology.     

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.     

Proteomic analysis of fatty-acylated proteins

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Proteomic analysis of SET-binding proteins

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2-DE and identified by MS using a MALDI-TOF. This experimental approach allowed the recognition of a number of SET-binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2alpha, glycogen phosphorylase (GP), and TCP1-beta) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein-protein interactions.     

Proteomic analysis of cartilage proteins

While the analysis of the cartilage proteome is important for our comprehensive understanding of the development and disease of this important tissue, several unique features of cartilage present some technical obstacles. Firstly, cartilage is difficult to obtain in adequate quantities for many protein analyses, especially from mice which are otherwise powerful experimental models. Furthermore, the cartilage extracellular matrix contains an insoluble network of collagen II-containing fibrils that are integrated within an abundant anionic network of aggrecan and hyaluronan aggregates. These interacting networks provide a structural scaffold for the covalent and non-covalent attachment of other proteins and glycoproteins. Consequently, proteomic analysis of cartilage requires extraction of proteins with chaotropic agents to achieve and significant protein solubilization. Finally, isolated chondrocytes are phenotypically unstable, which requires rapid isolation of cells or the use of specific culture conditions. Despite these problems, recent improvements in the sensitivity and reproducibility of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) techniques, combined with improved tissue preparation and sample pre-fractionation approaches, have made the proteomic characterization of cartilage tissues possible. Here we review the approaches that have been used and describe in detail protocols for the proteomic analysis of cartilage tissues and cells.     

The removal of exogenous thiols from proteins by centrifugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.     

Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

Background  

Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL), endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM) method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis.     

Separation of mammalian cell surface proteins by a two-dimensional gel electrophoresis system

Separation of externally exposed plasma membrane proteins of mammalian cells has been achieved by a new two-dimensional gel electrophoresis system. The proteins were separated in the first dimension on cylindrical polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) and in the second dimension on polyacrylamide slab gels containing 9 M urea, 0.1% SDS, and 0.1% Triton CF10. Using this method we have obtained reproducible high-resolution patterns of cell surface proteins of differentiated rat neuro-tumor cells in culture and of normal rat retinal cells. Different cell types show characteristic cell surface proteins in addition to ubiquitous ones. The number of common surface proteins between two cell types account for approximately half of the total surface proteins. By immunoprecipitation we have also found that rabbit anti-serum against a rat neuronal cell line can recognize most of these external proteins. Since the separation in the first dimension is done in the presence of SDS and the second dimension in the presence of SDS, a non-ionic detergent, and urea, the technique is particularly suitable for proteins that are of poor solubility. In addition to size, net charge and hydrophobicity appear to be important factors in the separation. Virtually all of the proteins that run in the first dimension can be recovered and further separated in the second.     

Interspecies immunologic cross-reactivity of mammalian sperm basic proteins

The species uniqueness of the sperm basic nuclear proteins (SBNPs) of mammals is in sharp contrast to the highly conserved function of those proteins, to mediate extreme condensation of the sperm chromatin. Despite their molecular uniqueness, however, the SBNPs of all eutherian mammals share certain characteristics: low molecular weight, high percentage of arginine and cysteine residues and, in the final step of chromatin condensation, extensive SS cross-linking. By radioimmunoassay, we have demonstrated that rabbit antiserum to mouse SBNP is reactive with purified mouse SBNP and mouse sperm and is cross-reactive with purified bull SBNP and sperm of bull, rat, rabbit, dog and man. The immunologically cross-reactive sites, therefore, may represent the homologous regions of the various species of SBNPs that serve the function of chromatin condensation. Immunologic reactivity was displayed only by sperm made to swell by treatment with 2-mercaptoethanol. Unswollen sperm were not reactive, confirming previous observations by immunofluorescence [1] indicating that reduction of SS bonds is necessary to render the antigenic sites of SBNP available for immunologic recognition, and suggesting that formation of SS bonds within that protein may be a molecular mechanism of antigen sequestration in vivo.     

DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins

Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [³²P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.     

Proteomic analysis of nucleoporin interacting proteins

The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex.     

Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.