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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们已经使用了一种无标记的定量纳米ESI-HPLC质谱学方法来 用比较蛋白质组学方法对正常大鼠和荷瘤大鼠的尿液进行分析 4-羟丁基(丁基)亚硝胺诱发的膀胱肿瘤。在这项研究中，尿液 来自对照动物(n=10)和可触及的膀胱肿瘤患者(n= 10)还原后依次用内切酶Lys C和胰酶消化 和烷基化反应。用纳米级反相高效液相色谱分离多肽， 用纳米螺旋电离，并用混合线性四极傅立叶分析 变换离子回旋质谱仪(FT-MS)获取了亲代质量扫描 对于20个样品中的每个样品，乙腈的线性梯度都超过2小时。质谱学 来自对照组和荷瘤组的个体进行了比对和准确 使用Rosetta ElucidatorTM软件对大量信号进行量化。同位素基团 两组间差异有统计学意义(P&lt；0.01) 用于产生精确的m/z值，用于定向采集串联质量 对多肽进行测序并确定两者之间的蛋白质差异的光谱 组。在携带肿瘤的尿液中发现增加的蛋白质中 (在排除肝脏衍生的和公认的血液蛋白例如白蛋白之后， Hbopexin、Hb)是Meprin、E-钙粘蛋白、透明质酸受体、糖蛋白 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.