Search for Novel Plasma Membrane Proteins as Potential Biomarkers in Human Mesenchymal Stem Cells Derived from Dental Pulp, Adipose Tissue, Bone Marrow, and Hair Follicle.

One of the drawbacks preventing the use of mesenchymal stem cells (MSCs) in clinical practice is the heterogeneous nature of their cultures. MSC cultures are not homogeneously formed by the MSCs and may contain non-mesenchymal cell types. Therefore, prior to use in clinics or research, complete characterization of MSCs should be performed to demonstrate the existence or absence of proper stem cell markers, many of which are happened to be cell-surface proteins. Unfortunately, the success of MSC characterization studies is limited due to the low specificity of the currently available cell-surface markers. Therefore, in this study, we aimed to investigate the plasma membrane (PM) proteins of MSCs isolated from human dental pulp (DP), adipose tissue (AT), bone marrow (BM), and hair follicle (HF) with the hope of proposing novel putative specific MSC markers. Differential-velocity centrifugation was used to enrich PM proteins. The isolated proteins were then identified by nLC-MS/MS and subjected to bioinformatics analysis. Proteins that were unique to each MSC type (CD9, CD10, CD63 for DP-MSCs; CD26, CD81, CD201, CD364 for AT-MSCs; Cd49a, CD49d for HF-MSCs; CD49e, CD56, CD92, CD97, CD156b, CD156c, CD220, CD221, CD298, CD315 for BM-MSCs) and common to all four MSC types (CD13, CD29, CD44, CD51, CD59, CD73, CD90) were identified. Uncharacterized proteins that have transmembrane (TM) domains were also detected. Some of the proteins identified in this study were the putative cell-surface markers that might be used for characterization of MSCs.

Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins.

Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.

Comparative Proteome Analysis of Breast Cancer Tissues Highlights the Importance of Glycerol-3-phosphate Dehydrogenase 1 and Monoacylglycerol Lipase in Breast Cancer Metabolism.

BACKGROUND/AIM: Breast cancer (BC) incidence and mortality rates have been increasing due to the lack of appropriate diagnostic tools for early detection. Proteomics-based studies may provide novel targets for early diagnosis and efficient treatment. The aim of this study was to investigate the global changes occurring in protein profiles in breast cancer tissues to discover potential diagnostic or prognostic biomarkers. MATERIALS AND METHODS: BC tissues and their corresponding healthy counterparts were collected, subtyped, and subjected to comparative proteomics analyses using two-dimensional gel electrophoresis (2-DE) and two-dimensional electrophoresis fluorescence difference gel (DIGE) coupled to matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/TOF) to explore BC metabolism at the proteome level. Western blot analysis was used to verify changes occurring at the protein levels. RESULTS: Bioinformatics analyses performed with differentially regulated proteins highlighted the changes occurring in triacylglyceride (TAG) metabolism, and directed our attention to TAG metabolism-associated proteins, namely glycerol-3-phosphate dehydrogenase 1 (GPD1) and monoacylglycerol lipase (MAGL). These proteins were down-regulated in tumor groups in comparison to controls. CONCLUSION: GPD1 and MAGL might be promising tissue-based protein biomarkers with a predictive potential for BC.

Response of Escherichia coli to Prolonged Berberine Exposure.

Berberine is a plant-derived alkaloid possessing antimicrobial activity; unfortunately, its efflux through multidrug resistance pumps reduces its efficacy. Cellular life span of Escherichia coli is generally shorter with prolonged berberine exposure; nevertheless, about 30% of the cells still remain robust following this treatment. To elucidate its mechanism of action and to identify proteins that could be involved in development of antimicrobial resistance, protein profiles of E. coli cells treated with berberine for 4.5 and 8 hours were compared with control cells. A total of 42 proteins were differentially expressed in cells treated with berberine for 8 hours when compared to control cells. In both 4.5 and 8 hours of berberine-treated cells, carbohydrate and peptide uptake regimens remained unchanged, although amino acid maintenance regimen switched from transport to synthesis. Defect in cell division persisted and this condition was confirmed by images obtained from scanning electron microscopy. Universal stress proteins were not involved in stress response. The significant increase in the abundance of elongation factors could suggest the involvement of these proteins in protection by exhibiting chaperone activities. Furthermore, the involvement of the outer membrane protein OmpW could receive special attention as a protein involved in response to antimicrobial agents, since the expression of only this porin protein was upregulated after 8 hours of exposure.