A proteomic approach for the identification of bismuth-binding proteins in Helicobacter pylori

Helicobacter pylori is a major human pathogen that can cause peptic ulcers and chronic gastritis. Bismuth-based triple or quadruple therapies are commonly recommended for the treatment of H. pylori infections. However, the molecular mechanisms underlying treatment with bismuth are currently not fully understood. We have conducted a detailed comparative proteomic analysis of H. pylori cells both before and after treatment with colloidal bismuth subcitrate (CBS). Eight proteins were found to be significantly upregulated or downregulated in the presence of CBS (20 μg mL⁻¹). Bismuth-induced oxidative stress was confirmed by detecting higher levels of lipid hydroperoxide (approximately 1.8 times) and hemin (approximately 3.4 times), in whole cell extracts of bismuth-treated H. pylori cells, compared with those from untreated cells. The presence of bismuth also led to an approximately eightfold decrease in cellular protease activities. Using immobilized-bismuth affinity chromatography, we isolated and subsequently identified seven bismuth-binding proteins from H. pylori cell extracts. The intracellular levels of four of these proteins (HspA, HspB, NapA and TsaA) were influenced by the addition of CBS, which strongly suggests that they interact directly with bismuth. The other bismuth-interacting proteins identified were two enzymes (fumarase and the urease subunit UreB), and a translational factor (Ef-Tu). Our data suggest that the inhibition of proteases, modulation of cellular oxidative stress and interference with nickel homeostasis may be key processes underlying the molecular mechanism of bismuth’s actions against H. pylori. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu⁴⁺-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

A shotgun proteomic method for the identification of membrane-embedded proteins and peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.     

A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides

Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.     

A proteomic approach for identification and localization of the pericellular components of chondrocytes

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.     

A multidimensional proteomic approach to identify hypertrophy-associated proteins

Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.     

A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus

The saliva of ticks contains anti-haemostatic, anti-inflammatory and immunomodulatory molecules that allow these parasites to obtain a blood meal from the host and help tick-borne pathogens to infect the vertebrate host more efficiently. This makes the salivary molecules attractive targets to control ticks and tick-borne pathogens. Although Ornithodoros moubata and O. erraticus are important argasid ticks that transmit severe diseases, to date only a few of their salivary proteins have been identified. Here we report our initial studies using proteomic approaches to characterize the protein profiles of salivary gland extracts (SGE) from these two argasids. The present work describes the proteome of the SGEs of both tick species, their antigenic spots, and the identification of several of their proteins. The whole number of identifications was low despite the good general quality of the peptide mass maps obtained. In the O. moubata SGE, 18 isoforms of a protein similar to O. savignyi TSGP1 were identified. In the O. erraticus SGE we identified 6 novel proteins similar to unknown secreted protein DS-1 precursor, NADPH dehydrogenase subunit 5, proteasome alpha subunit, ATP synthase F0 subunit 6, lipocalin and alpha tubulin. Finally, the current drawbacks of proteomics when applied to the identification of acarine peptides and proteins are discussed.     

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.     

A target-specific approach for the identification of tyrosine-sulfated hemostatic proteins

A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [³⁵S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [³⁵S]sulfate-labeled hemostatic proteins, and tyrosine O-[³⁵S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins—protein S, prekallikrein, and plasminogen—were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.     

Identification of disease-associated proteins by proteomic approach in ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic systemic inflammatory disorder of the axial skeleton and shows significant inherited susceptibility. Auto-immune responses have been traditionally considered as a putative pathogenetic event in AS. However, no consistent self-antigen has been identified to responsible for the disorders in AS to this day. In this study, serum protein profiles of AS patients and healthy controls from a large Chinese AS family were investigated by two dimensional electrophoresis analysis. A group of four highly expressed protein spots was observed in all AS patients' profiles and subsequently identified as isoforms of haptoglobin precursor (pre-Hp) by ESI-Q-TOF MS/MS. Increased expression of haptoglobin precursor were also observed in sera of sporadic AS patients. Moreover, bioinformatics analysis revealed epitopes derived from haptoglobin precursor with high affinity binding to HLA-B( *)2705, a primary subtype associated with AS. These results indicate that pre-Hp may be involved in the pathogenesis of AS.     

Mechanism of autodegradation of cell-surface macromolecules shed by human melanoma cells

The mechanism of autodegradation of cell-surface macromolecules shed by human melanoma cells was studied by incubating radio-iodinated shed macromolecules with unlabeled sister cells and measuring the appearance of acid-soluble radioactivity. After a preliminary latent period of 1-3 h, degradation continually increased up to 24 h and was concentration-dependent. By contrast, binding to cells was very rapid reaching half-maximal value within 15 min. Autodegradation was markedly reduced (44-82%) by pharmacological agents which interfere with endocytosis or lysosomal enzyme activity, including drugs which inhibit receptor migration into coated pits (dansylcadaverine), endocytosis and intracellular transport (colchicine, cytochalasin B, and monensin), and the activity of lysosomal enzymes (chloroquine, ammonium chloride, leupeptin). Degradation was almost totally suppressed (95%) at 4 degrees C. These data suggest that surface macromolecules shed by melanoma cells are autodegraded in part by re-uptake into melanoma cells followed by degradation in lysosomes.     

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.     

A proteomic approach to analyze salt-responsive proteins in rice leaf sheath

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to 3-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY363) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (SOD). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, SOD maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of SOD was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY363 and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY363 was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.     

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.     

A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein,Frigg, as a protein kinase A substrate

Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways.     

A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

A proteomic approach to the identification of new tPA receptors in pancreatic cancer cells

We have developed a strategy to identify putative tissue-type plasminogen activator (tPA)receptors present in pancreatic cancer cells by affinity capture with tPA-Sepharose followed by 2-DE and MALDI-MS PMF. Proteins pulled down from either total lysates or raft membrane fractions were characterized and compared with those from a total lysate of an endothelial cell line (HUVEC) to identify pancreas-restricted tPA receptors. A total of 31 proteins were found by this approach, including annexin A2, already described as a tPA receptor in pancreas and endothelial cells, other proteins acting as tPA receptors (i.e., enolase, cytokeratins 8 and 18) in other tissues, and additional proteins not previously identified as candidate tPA receptors. Confirmation of the results was performed for some of these proteins using immunoblotting. These studies are the basis for further functional analyses on the role of these proteins in the biological effects of tPA.