DEVELOPMENT OF A PROTEOMIC STRATEGY FOR ANALYSIS OF PLASMA MEMBRANE PROTEINS

Plasma membrane (PM) proteins play crucial roles in cell signaling and communications, and they are the targets of more than two thirds of drugs currently under development. Studies on changes in protein content, quantity and modifications of the PM proteins indicate metabolic alteration of disease related cells; therefore, mass spectrometry-based proteomic studies may lead to improved understanding of the pathology, the characterization of novel biomarkers, and discovery of future drug targets. The main objectives of my research are to develop an effective enrichment strategy and to optimize the proteomic workflow for analysis of PM proteins from cells in suspension. Strategies were optimized with human multiple myeloma cells cultured in suspension, and optimized strategies were applied to study the PM proteome of myeloid-derived suppressor cells (MDSC) collected from an animal model. We focus on optimization of the cationic nanoparticle pellicle method for enrichment of PM proteins. The principle of this method is to attach cationic nanoparticles to the cell surface by electrostatic interaction between the positively charged nanoparticles and the negatively charged cell surface. Thus, the heavier coated-plasma membrane sheets can be separated more easily from cellular organelles by centrifugation after cell lysis. The isolated PM proteins were identified by LC-MS/MS analysis. We have also optimized the workflow for proteolysis to enhance identification of hydrophobic PM proteins. Our studies reveal that higher density nanoparticle pellicles provide higher enrichment efficiency of the PM proteins and that a procedure using digestion in the gel matrix enhances the analysis of highly hydrophobic proteins. The most effective enrichment technique and optimized proteomic procedures were applied to characterize the PM proteins from MDSC obtained from BALB/c mice carrying 4T1 mammary carcinomas. These cells are known to accumulate in individuals with cancer and suppress anti-tumor immunity. Their accumulation and activity are increased with heightened-levels of inflammation. Comparative studies of the PM proteins expressed in the cells derived from basal- and heightened- levels of inflammation were performed using the spectral counting method. This work reveals a set of protein candidates that have a high potential to be involved in the inflammation-driven immunosuppressive activity of the MDSC

Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells

An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NPbacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NPcell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, e.g., via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, e.g., by SEM, for evidence of NPbacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NPbacteria interactions.financial support from the following sources: grant SFRH/BPD/47693/2008 from the Portuguese Foundation for Science and Technology (FCT); FCT Strategic Project PEst-OE/EQB/LA0023/2013; project “BioHealth Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2−O Novo Norte), QREN, FEDER; project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, ref. FCOMP-01-0124-FEDER- 027462

Watermelon seeds and peels: fatty acid composition and cosmeceutical potential

Watermelon consumption results in generation of organic waste in the form of seeds and peels. We have evaluated the fatty acid profiles and antioxidant content of watermelon (Kinnaree cultivar) seed oil and peel wax. In addition, we assessed the potential use of these watermelon industry byproducts in the development of cosmeceuticals. The most abundant fatty acids in seed oil and peel wax were linoleic acid and arachidic acid, respectively. Fatty acids form an essential component in the cell membranes and have seen increased recognition in the cosmeceutical industry. Antioxidants also play a beneficial role in skincare in combating free-radicals resulting from sun damage and pollutants. The seed oil showed stronger antioxidant activity than the peel wax, as indicated by the DPPH radical scavenging ability of 0.894 mg α-tocopherol equivalent/g dried seeds versus 0.036 mg α-tocopherol equivalent/g dried peels. Therefore, the seed oil was formulated into skincare products, in the form of emulsions and nanoemulsions. The most effective formulae were stable at room temperature for seven days, or following repeated cycles of heating and cooling. This work demonstrates the potential for watermelon seed oil to be employed in skincare product formulations, which could maximize agricultural profit and minimize environmental waste

Exosomes from Myeloid-Derived Suppressor Cells Carry Biologically Active Proteins

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages toward a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered >2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages toward a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes

Comparative Study of Workflows Optimized for In-gel, In-solution, and On-filter Proteolysis in the Analysis of Plasma Membrane Proteins

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins

Enrichment of Plasma Membrane Proteins Using Nanoparticle Pellicles: Comparison between Silica and Higher Density Nanoparticles

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins

Enrichment of Plasma Membrane Proteins Using Nanoparticle Pellicles: Comparison between Silica and Higher Density Nanoparticles

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins