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    Comparative proteomics analysis of four commonly used methods for identification of novel plasma membrane proteins
    (Springer, 2019) Yöneten, Kübra Karaosmanoğlu; Kasap, Murat; Akpınar, Gürler; Kanlı, Aylin; Karaöz, Erdal
    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.
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    Effects of Lucilia sericata on wound healing in streptozotocin-induced diabetic rats and analysis of its secretome at the proteome level
    (Sage Publications Ltd, 2018) Tombultürk, Fatma Kübra; Kasap, Murat; Tunçdemir, Matem; Polat, Erdal; Sirekbasan, Serhat; Kanlı, Ali İsmet; Kanıgür-Sultuybek, Gönül
    The use of Lucilia sericata larvae on the healing of wounds in diabetics has been reported. However, the role of the excretion/secretion (ES) products of the larvae in treatment of diabetic wounds remains unknown. This study investigated whether application of the ES products of L. sericata on the wound surface could improve the impaired wound healing in streptozotocin-induced diabetic rats. Additional analysis was performed to understand proteome content of L. sericata secretome to understand ES contribution at the molecular level. For this purpose, full-thickness skin wounds were created on the backs of diabetic and control rats. A study was conducted to assess the levels of the ES-induced collagen I/III expression and to assay nuclear factor B (NF-B) (p65) activity in wound biopsies and ES-treated wounds of diabetic rat skin in comparison to the controls. The expression levels of collagen I/III and NF-B (p65) activity were determined at days 3, 7, and 14 after wounding using immunohistological analyses and enzyme-linked immunosorbent assay technique. The results indicated that treatment with the ES extract increased collagen I expressions of the wound control and diabetic tissue. But the increase in collagen I expression in the controls was higher than the one in the diabetics. NF-B (p65) activity was also increased in diabetic wounds compared to the controls, whereas it was decreased in third and seventh days upon ES treatment. The results indicated that ES products of L. sericata may enhance the process of wound healing by influencing phases such as inflammation, NF-B (p65) activity, collagen synthesis, and wound contraction. These findings may provide new insights into understanding of therapeutic potential of ES in wound healing in diabetics.
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    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
    (Springer Link, 2021) Akpınar, Gürler; Karaosmanoğlu Yoneten, Kübra; Kasap, Murat; Karaöz, Erdal
    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.