TARGETED, LABEL-FREE PROTEOMIC ANALYSIS OF URINE IN A RAT BLADDER CANCER MODEL

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 我们使用了无标记的定量nanoESI-HPLC质谱法， 对对照组和荷瘤大鼠的尿液进行比较蛋白质组学分析， 4-羟丁基亚硝胺诱发膀胱肿瘤。在这项研究中， 来自对照动物（n = 10）和来自具有可触知膀胱肿瘤的动物（n = 10）的蛋白质。 10)还原后依次用内切蛋白酶Lys C和胰蛋白酶消化 和烷基化。用纳流-反相HPLC分离肽， 用纳米喷雾离子化进行离子化，并用混合线性四极傅立叶分析仪进行分析。 变换离子回旋质谱仪（FT-MS）。 获得母体肿块扫描 对于20份样品中的每一份，采用2小时线性梯度的乙腈进行检测。 质谱 将对照组和荷瘤组的个体进行比对， 使用RosettaElucidatorTM软件定量质量信号。同位素组 两组间差异有统计学意义（P < 0.01）的面积为 用于产生精确的m/z值，用于直接采集串联质量 光谱来对肽进行测序并鉴定两者之间的蛋白质差异 组在携带肿瘤的尿液中发现增加的蛋白质中， （在排除肝来源的和公认的血液蛋白例如白蛋白之后， 血红素结合蛋白，血红蛋白）是甲氧苄氨嘧啶，E-钙粘蛋白，透明质酸受体，glycam， 视网膜母细胞瘤结合蛋白

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have used a label-free quantitative nanoESI-HPLC mass spectrometric method to perform comparative proteomics analysis on the urine from control rats and rats bearing 4-hydroxybutyl(butyl)nitrosamine induced bladder tumors. In this study the urinary proteins from control animals (n = 10) and from those with palpable bladder tumors (n = 10) were digested sequentially with endoprotease Lys C and trypsin after reduction and alkylation. The peptides were separated with nanoflow- reversed phase HPLC, ionized with nanospray ionization, and analyzed with a hybrid linear quadrupole Fourier transform ion cyclotron mass spectrometer (FT-MS). Parent mass scans were acquired over a 2 h linear gradient of acetonitrile for each of the 20 samples. The mass spectra from the control and tumor-bearing groups were aligned and the individual accurate mass signals were quantified using Rosetta ElucidatorTM software. The isotope groups with statistically significant different areas (P < 0.01) between the two groups were used to produce the accurate m/z values for directed acquisition of tandem mass spectra to sequence the peptide and identify the protein differences between the two groups. Among the proteins that were found to be increased in the tumor bearing urine (after excluding the hepatic-derived and well-recognized blood proteins e.g. albumin, hemopexin, hemoglobin) were meprin, E-cadherin, hyaluronan receptor, glycam, retinoblastoma binding protein.