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 453 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.
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PMID:Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry. 1093 58

Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification. We applied this procedure to select and identify triplex-binding activities in HeLa nuclear extracts. To achieve this, we used a panel of deoxyribonucleic probes adopting intramolecular triple-helices, varying in their primary sequence, structure or triple-helix motif. A limited number of spots was reproducibly revealed by South-western blotting. Spots of interest were localised among a complex population of (35)S-labelled proteins according to their (32)P-specific emission. Position of the same spots was extrapolated on a preparative gel coloured with Coomassie blue, allowing excision and purification of the corresponding proteins. The material was subjected to mass spectrometry upon trypsin digestion and MALDI-TOF peptide fingerprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, E1 and I). The identities of several of them were confirmed by comparing western and South-western blots on the same membrane using specific antibodies. The recognition specificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in different contexts: single strand to a small extent, triplex and possibly other higher-order structures.
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PMID:Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry. 1137 62

The hepatotoxicity of bromobenzene is strongly correlated with the covalent binding of chemically reactive metabolites to cellular proteins, but up to now relatively few hepatic protein targets of these reactive metabolites have been identified. To identify additional hepatic protein targets we injected an hepatotoxic dose of [14C]bromobenzene to phenobarbital-pretreated male Sprague-Dawley rats ip. After 4 h, their livers were removed and homogenized, and the homogenates fractionated by differential ultracentrifugation. The highest specific radiolabeling (6.1 nmol equiv 14C/mg of protein) was observed in a particulate fraction (P25) sedimented at 25000g from a 6000g supernatant fraction. Proteins in this fraction were separated by two-dimensional electrophoresis and, after transblotting, analyzed for radioactivity by phosphorimaging. More than 20 radiolabeled protein spots were observed in the blots. For 17 of these spots, peptide mass maps were obtained using in-gel digestion with trypsin, followed by MALDI-TOF mass spectrometric analysis of the resulting peptide mixtures. By searching genomic databases, the 17 sets of MS-derived peptide masses were found to match predicted tryptic fragments of just 7 proteins. Spots 1-4 matched with 78 kDa glucose regulated protein (GRP78), protein disulfide isomerase isozyme A1 (PDIA1), endoplasmic reticulum protein ERp29, and PDIA6, respectively. Spots 5 and 6, 7-11, and 12-17 presented as apparent "charge trains" of spots, each of which gave peptide mixtures closely similar to those of other spots within the train. The proteins present in these sets of spots were identified as transthyretin, serum albumin precursor and PDIA3, respectively. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.
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PMID:Identification of seven proteins in the endoplasmic reticulum as targets for reactive metabolites of bromobenzene. 1201 92

Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.
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PMID:Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis. 1281 46

Partially purified transducin was resolved using two-dimensional gel electrophoresis (2-DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa beta-subunit of transducin (T(beta)) was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. As many as six spots with different pI, ranging from 5.2 to 6.1, were observed when separated using 2-DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide-binding protein G(I)/G(S)/G(T) beta-subunit 1 (GNB1; T(beta1)). This suggested that post-translational modification was responsible for the differences in pI. Phosphorylation experiments showed that at least one T(beta1) spot was phosphorylated in vitro with [gamma-(32)P]ATP by an endogenous kinase. Treatment of T(beta) with alkaline phosphatase caused a large change in the spot pattern of T(beta), suggesting that phosphorylated T(beta) is a substrate for alkaline phosphatase. We conclude that T(beta1) constitutes over 99% of the T(beta) expressed in bovine rod outer segments and displays structural heterogeneity that is due to post-translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1.
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PMID:Proteomic analysis of transducin beta-subunit structural heterogeneity. 1459 96

The phloem transport system is a complex tissue that primarily carries photoassimilate from source to sink. Its function depends on anucleate sieve elements (SE) supported by companion cells (CC). In this study, SE sap was sampled and the protein identity of soluble proteins was determined with the aim of understanding the function of proteins within the conduit. Unlike many plants, SE sap exudes from incisions in the bark of Ricinus communis and, although there is a greater possibility of contamination from tissues other than SE, sap can be obtained in sufficient quantities to separate proteins using 2D electrophoresis. Spots were excised for trypsin digest, then analysed by quadrupole time of flight (Q-TOF) mass spectrometry (MS) and database searched to determine sequence identity. Overall, 18 proteins were identified in the SE-enriched sap. Proteins identified that have not previously been identified directly from SE sap included a glycine-rich RNA-binding protein, metallothionein, phosphoglycerate mutase, and phosphopyruvate hydratase. The potential role of the identified protein in SE function is discussed. The protein identification in this study provides a first step towards the goal of a greater understanding of the function of proteins within the SE.
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PMID:Determining protein identity from sieve element sap in Ricinus communis L. by quadrupole time of flight (Q-TOF) mass spectrometry. 1518 Nov 2

Hindlimb unloading has been used as a model for bone loss associated with extended bed rest or space travel. However, this model also reduces muscle mass and influences other biological systems. To evaluate the impact of hindlimb unloading on bone and overall health, we applied 2-D gel electrophoresis (2-DE)-based proteomics to serum samples collected from 24 5-month-old female rats that were treated for 2 weeks under three conditions: control, hindlimb unloading (HU) and unloading plus bisphosphonate (HUA) (n=8/group). Prior to the intervention, rats were injected with 3H-tetracycline to label bone surfaces. At the end of the experiment bone, urine, and serum samples were collected. As expected, HU reduced femur aBMD and BMC and increased daily urinary 3H-tetracycline (a measure of bone resorption rate) and these effects were reversed by bisphosphonate. In addition, serum osteocalcin and TRAP5b were decreased in the HUA compared to control and HU. Abundant proteins, albumin, IgG and transferrin were removed from serum samples prior to 2-DE analysis (n=5 analytical replicates). Statistical analysis of spot intensities revealed 53 differentially expressed spots among the 3 groups. Cluster analysis shows that 30 spots reflect changes unique to the HU group (i.e. potential bone biomarkers), 6 unique to HUA (i.e. drug related), and 17 common to HU and HUA (e.g. potential mental stress or muscle loss markers). Spots were identified by LC-MS/MS after in-gel trypsin digestion and were found to relate to a variety of physiological functions.
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PMID:Effects of hindlimb unloading and bisphosphonates on the serum proteome of rats. 1762 11

During coffee seed development, proteins are predominantly deposited in cotyledons and in the endosperm. Reserve proteins of the 11S family are the most abundant globulins in coffee seeds, acting as a nitrogen source during roasting and guaranteeing flavor and aroma. The aim of the present study was to compare the protein profiles of endosperm and zygotic embryos of coffee seeds. Proteins were extracted from whole seed as well as from embryo and endosperm, separately. Total proteins were analyzed by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MS). The most abundant spots observed in the gels of coffee seeds were excised, digested with trypsin, and identified by MS as subunits of the 11S globulin. Spots with identical pI and molecular masses were also observed in the protein profiles of coffee endosperm and embryo, indicating that 11S protein is also highly expressed in those tissues. Peptide sequence coverage of about 20% of the entire 11S globulin was obtained. Three other proteins were identified in the embryo and endosperm 2-DE profiles as a Cupin superfamily protein, an allergenic protein (Pru ar 1), exclusive to the endosperm 2D map, and a hypothetical protein, observed only in the zygotic embryo profile.
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PMID:Comparative proteomical analysis of zygotic embryo and endosperm from Coffea arabica seeds. 1895 16

Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10(-7) mol l(-1)). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of +/-1.5 were robotically picked for in-gel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK-III reduces the cytosolic presence of subunits A and B of the V-type H(+) ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca(2+)-binding protein regucalcin/SMP30 and actin-depolymerizing factor. Supporting the putative role of PKA in the AK-III-induced activation of the V-type H(+) ATPase is the effect of H89, an inhibitor of PKA, on fluid secretion. H89 reverses the stimulatory effect of AK-III on transepithelial fluid secretion in isolated Malpighian tubules. However, AK-III does not raise intracellular levels of cAMP, the usual activator of PKA, suggesting a cAMP-independent activation of PKA that removes subunits A and B from the cytoplasm in the assembly and activation of the V-type H(+) ATPase. Alternatively, protein kinase C could also mediate the activation of the proton pump. Ca(2+) remains the primary intracellular messenger of the aedeskinins that signals the remodeling of the paracellular complex apparently through protein kinase C, thereby increasing transepithelial anion secretion. The effects of AK-III on active transcellular and passive paracellular transport are additive, if not synergistic, to bring about the rapid diuresis.
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PMID:Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules. 1915 Dec 7

Royal jelly is widely consumed in the community and has perceived benefits ranging from promoting growth in children and improvement of general health status to enhancement of longevity for the elderly. However, royal jelly consumption has been linked to contact dermatitis, acute asthma, anaphylaxis and death. High prevalence of positive skin tests to royal jelly have been reported among atopic populations in countries with a high rate of royal jelly consumption. The present study is aimed to identify the major allergens of royal jelly. Royal jelly extract was separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional electrophoresis (2-D). Immunoblotting of the SDS-PAGE and 2-D profiles were performed to identify the allergenic spots. Spots were then excised from the 2-D gel, digested with trypsin and analyzed by mass spectrometry. The SDS-PAGE of royal jelly extract revealed 18 bands between 10 to 167 kD. Western blot of the fractionated proteins detected 15 IgE-binding bands between 14 to 127 kD with seven major allergens of 32, 40, 42, 49, 55, 60 and 67 kD using serum from 53 subjects with royal jelly allergy. The 2-D gel fractionated the royal jelly proteins to more than 50 different protein spots. Out of these, 30 spots demonstrated specific IgE affinity to the sera tested. Eight spots of the major royal jelly allergens were selected for mass-spectrometry analysis. Digested tryptic peptides of the spots were compared to the amino acid sequence search in protein databases which identified the fragments of royal jelly homologus to major royal jelly protein 1 (MRJ1) and major royal jelly protein 2 (MRJ2). In conclusion, the major allergens of royal jelly are MRJ1 and MRJ2 in our patients' population.
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PMID:Characterization of major allergens of royal jelly Apis mellifera. 1928 64


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