Proteomic analysis of lifestyle related diseases

在过去的几年里，通过寻找生活方式相关疾病的生物标志物，探索生物体液蛋白质组的兴趣已经扩大。在本研究中，我们研究了代谢综合征动物模型SHR/NDmcr-cp大鼠（SHR/NDmcr-cp）胰腺的蛋白质组学。取20周龄雄性SHR/NDmcr-cp大鼠和Wistar-Kyoto/Izm （WKY/Izm）正常大鼠胰腺组织。这两个样品（1 g湿重）用10 mL裂解缓冲液均质，其中含有7 M尿素，4% 3-[（胆酰胺丙基）二甲基氨]-1-丙磺酸，2 M硫脲和5 mM醋酸镁/ 30 mM Tris-HC1缓冲液（pH 8.0）。在40℃下400,000 x g离心60分钟后，上清液用于二维电泳（2-DE）样品。将100 μg蛋白用补液稀释至350 μL，涂于Immobiline干条上（线性梯度在pH 3至10,13 cm之间（GE Healthcare biosciences， NJ， USA））。更多的研究集中在IPG-phor系统（GE Healthcare biosciences）上。第一维电泳后，将条带在含二硫苏糖醇的溶液中平衡15分钟，用碘乙酰胺取代二硫苏糖醇。在SE 600 Ruby （GE Healthcare biosciences）上使用12.5% sds -聚丙烯酰胺凝胶进行二维电泳分离。用2-DE凝胶分离胰腺蛋白，用考马斯亮蓝染色。SHR/NDmcr-cp样品在2-DE凝胶上的32个斑点比WKY/Izm样品染色更多。然后将这32个斑点切开，用含有胰蛋白酶的消化缓冲液浸泡。蛋白消化过夜后，肽片段用Zip tips C18脱盐，提取后用AXIMA-CFR + MALDI TOF-MS分析。随后，利用Swiss-Prot和ncbin数据库（仅限于啮齿类动物），通过MASCOT程序对该蛋白进行了鉴定。有10种蛋白质与数据库中的蛋白质显著匹配。这些蛋白包括a-烯醇化酶、磷酸甘油酸激酶、支链氨基酸转移酶、羧肽酶Al前体、α-蛋白、肌醇单磷酸酶乳糜蛋白酶原B前体、次黄嘌呤-鸟嘌呤磷酸核糖基转移酶、核苷二磷酸激酶B、弹性酶2前体和膜联蛋白A5。为了寻找生活方式相关疾病的生物标志物，代谢综合征动物模型血浆蛋白质组的进一步研究正在进行中。少

In the last few years, the interest in exploring the proteome of biological fluids has expanded through the search for biomarkers of lifestyle related diseases. In this study, we investigated the proteome of pancreas in an animal model of metabolic syndrome, SHR/NDmcr-cp rat (SHR/NDmcr-cp). The pancreas tissues extirpated from twenty-week-old male SHR/NDmcr-cp and normative rats, Wistar-Kyoto/Izm (WKY/Izm). These two samples (1 g wet weight) were homogenized with 10 mL of a lysis buffer, containing of 7 M urea, 4% 3-[(cholamidopropyl) dimethyl ammonio]-1-propanesulfonate, 2 M thiourea, and 5 mM magnesium acetate / 30 mM Tris-HC1 buffer (pH 8.0). After centrifugation at 400,000 x g for 60 min at 4 0C, the supernatant was used for a two-dimensional electrophoresis (2-DE) sample. One hundred μg of a protein was diluted to 350 μL with a rehydration solution and applied onto Immobiline dry strips (linear gradient between pH 3 and 10, 13 cm (GE Healthcare Bio-sciences, NJ, USA)). The protein … More s were focused on a IPG-phor system (GE Healthcare Bio-sciences). After the first dimension electrophoresis, the strips were equilibrated for 15 min in a solution containing dithiothreitol, which was replaced by iodoacetamide. Separation in the second dimensional electrophoresis was carried out on a SE 600 Ruby (GE Healthcare Bio-sciences) using a 12.5% SDS-polyacrylamide gel. Pancreas proteins separated on a 2-DE gel were stained with coomassie brilliant blue. The 32 spots of the SHR/NDmcr-cp sample on the 2-DE gel were more stained than those of the WKY/Izm sample. Then those 32 spots were cut, and soaked with digestion buffer containing trypsin. After the overnight protein digestion, peptide fragments were desalted by Zip tips C18, extracted and analyzed with AXIMA-CFR plus MALDI TOF-MS. Thereafter, the protein was identified by a MASCOT program using Swiss-Prot and NCBInr databases limited to Rodents species. Ten proteins provided a significant match with databases' proteins. Those proteins included a-enolase, phosphoglycerate kinase, branched-chain-amino acid transferase, carboxy-peptidase Al precursor, α-protein, inositol monophosphatase chymotrypsinogen B precursor, hypoxanthine-guanine phosphoribosyltransferase, nucleoside diphosphate kinase B, elastase-2 precursor and annexin A5. Further study for the proteome of plasma in an animal model of metabolic syndrome is ongoing in order to search for biomarkers of lifestyle related diseases. Less