室温放置4 h对样本尿蛋白质组分析的影响

Effects of a 4-hour staying at room temperature on urinary proteome analysis

目的探讨用医院检验科尿常规检查剩余的尿液样本开展尿蛋白质组分析,以便充分利用资源。方法同一个体尿液样本采集后分别采用立即冻存和室温下放置4h后冻存于-80℃备用。尿液样本分别进行聚丙烯酰胺凝胶电泳、二维凝胶电泳和液相色谱串联质谱分析,对比两种尿液样本采集方法对尿蛋白质组分析的影响。用点杂交免疫印迹法分析了室温放置4h尿液样本(n=150)中胞质非特异性二肽酶(CNDP2)蛋白在结直肠癌(CRC)患者与健康对照尿液中的含量。结果立即冻存和经室温放置4h后冻存的尿液样本的主要蛋白条带、数量和位置无明显差异;从中分别鉴定了(1204±50,n=3)个和(1155±7,n=3)个尿蛋白质,以及(7501±661,n=3)条和(6940±182,n=3)条多肽,两组样本间无差异。尿蛋白CNDP2在结直肠癌患者尿液中含量显著高于健康对照(P<0.001)。结论采集后立即冻存和室温放置4h后冻存尿液样本的尿蛋白质组分析无明显差异。医院检验科尿常规检查剩余的尿液样本可用于尿蛋白质组分析。CNDP2在结直肠癌患者尿液中含量升高,是结直肠癌候选的尿蛋白标志物。

Objective Using the remainder urine samples of hospital laboratories for urinary proteomic analysis to save resource for urine sample collection.Methods Urine samples collected from the same individuals were divided into two parts.In which,half of them was frozen in-80℃immediately and the rest was kept in room temperature for 4 h(4 h-RT)before been frozen in-80℃.Samples were then analyzed by proteomics strategy including sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),two-dimensional gel electrophoresis(2-DE)and liquid chromatography tanden mass spectrometry(LC-MS/MS).The differentially expressed urinary proteins were then compared between the two sample collection methods.Using the urine samples(n=150)collected by 4 h-RT,the expression level of cytosolic non-specific dipeptidase 2(CNDP2)between colorectal cancer(CRC)patients and health controls was analyzed by Dot blot assay.Results There was no significant difference in the main protein bands,numbers and positions of SDS-PAGE and 2 DE analysis between the frozen immediately and 4 h-RT samples.A total of(1 204±50,n=3)kinds and(1 155±7,n=3)kinds of urine proteins,(7 501±661,n=3)and(6 940±182,n=3)peptides was identified with no significant difference between the two groups.The level of CNDP2 was significantly higher in the urine of CRC patients than that from healthy controls(P<0.001).Conclusions There is no significant difference in urinary proteome between the samples frozen immediately or 4 h-RT staying after collection.The urine samples after laboratory examinations in hospital can be used for urinary proteomics analysis.The expression level of CNDP2 is elevated in the urine of CRC patients and it may be a candidate marker of CRC.