Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

Proteomic analysis of microvesicles derived from human colorectal cancer cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.     

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

ABSTRACT: Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Characterization of three membrane fractions isolated from cells of Rhodopseudomonas capsulata adapting from chemotrophic to phototrophic conditions

By means of sucrose density centrifugation three membrane fractions, named light, medium and heavy have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Proteomic analysis of lamellar bodies isolated from rat lungs

Background  

Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.     

Adenylate cyclase activity of membrane fractions isolated from sea urchin sperm

The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I¹²⁵-labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15-fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg⁺⁺ and Na⁺ stimulated their cyclase activity about sevenfold at 2.5 mM Mn⁺⁺ and 3.2 mM ATP. At this ATP to Mn⁺⁺ ratio, high concentrations of Ca⁺⁺ have a small stimulatory effect.     

Proteomic analysis of plasma membrane lipid rafts of HL-60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.     

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.     

Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodopseudomonas capsulata.

Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.     

Proteomic analysis of lipid raft-enriched membranes isolated from internal organelles

The mitochondria-associated membrane (MAM) is a sub-region of the endoplasmic reticulum (ER) that facilitates crosstalk between the ER and mitochondria. The MAM actively influences vital cellular processes including Ca²⁺ signaling and protein folding. Detergent-resistant microdomains (DRMs) may localize proteins to the mitochondria/MAM interface to coordinate these events. However, the protein composition of DRMs isolated from this region is not known. Lipid-raft enriched DRMs were isolated from a combined mitochondria/MAM sample and analyzed using two-dimensional reversed-phased tandem mass spectrometry. Strict post-acquisition filtering of the acquired data led to the confident identification 250 DRM proteins. The majority (58%) of the identified proteins are bona fide mitochondrial or ER proteins according to Gene Ontology annotation. Additionally, 74% of the proteins have previously been noted as MAM-resident or -associated proteins. Furthermore, ∼20% of the identified proteins have a documented association with lipid rafts. Most importantly, known internal LR marker proteins (inositol 1,4,5-trisphosphate receptor type 3, erlin-2, and voltage-dependent anion channel 1) were detected as well as most of the components of the mitochondrial/MAM-localized Ca²⁺ signaling complex. Our study provides the basis for future work probing how the protein activities at the mitochondrion/MAM interface are dependent upon the integrity of these internal lipid-raft-like domains.     

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria.     

Proteomic analysis of microparticles isolated from malaria positive blood samples

Background

Malaria continues to be a great public health concern due to the significant mortality and morbidity associated with the disease especially in developing countries. Microparticles (MPs), also called plasma membrane derived extracellular vesicles (PMEVs) are subcellular structures that are generated when they bud off the plasma membrane. They can be found in healthy individuals but the numbers tend to increase in pathological conditions including malaria. Although, various studies have been carried out on the protein content of specific cellular derived MPs, there seems to be paucity of information on the protein content of circulating MPs in malaria and their association with the various signs and symptoms of the disease. The aim of this study was therefore to carry out proteomic analyses of MPs isolated from malaria positive samples and compare them with proteins of MPs from malaria parasite culture supernatant and healthy controls in order to ascertain the role of MPs in malaria infection.

Methods

Plasma samples were obtained from forty-three (43) malaria diagnosed patients (cases) and ten (10) healthy individuals (controls). Malaria parasite culture supernatant was obtained from our laboratory and MPs were isolated from them and confirmed using flow cytometry. 2D LC-MS was done to obtain their protein content. Resultant data were analyzed using SPSS Ver. 21.0 statistical software, Kruskal Wallis test and Spearman’s correlation coefficient r.

Results

In all, 1806 proteins were isolated from the samples. The MPs from malaria positive samples recorded 1729 proteins, those from culture supernatant were 333 while the control samples recorded 234 proteins. The mean number of proteins in MPs of malaria positive samples was significantly higher than that in the control samples. Significantly, higher quantities of haemoglobin subunits were seen in MPs from malaria samples and culture supernatant compared to control samples.

Conclusion

A great number of proteins were observed to be carried in the microparticles (MPs) from malaria samples and culture supernatant compared to controls. The greater loss of haemoglobin from erythrocytes via MPs from malaria patients could serve as the initiation and progression of anaemia in P.falciparum infection. Also while some proteins were upregulated in circulating MPs in malaria samples, others were down regulated.

     

Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.     

Proteomic analysis of a membrane skeleton fraction from human liver

The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.     

Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, constitutively secretes outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress has revealed that OMVs are essential for pathogenesis of P. aeruginosa, their proteins have not been comprehensively analyzed so far. In this study, we identified 338 vesicular proteins with high confidence by five separate LC-MS/MS analyses. This global proteome profile provides a basis for future studies to elucidate the pathological functions of OMVs from P. aeruginosa.     

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.     

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.