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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们使用了无标记的定量nanoESI-HPLC质谱法对对照大鼠和4-羟基丁基（丁基）亚硝胺诱导的膀胱肿瘤大鼠的尿液进行比较蛋白质组学分析。在本研究中，将对照动物（n = 10）和可触及膀胱肿瘤动物（n = 10）的尿蛋白在还原和烷基化后依次用内切蛋白酶Lys C和胰蛋白酶消化。用纳流-反相HPLC分离肽，用纳喷雾电离电离，并用混合线性四极傅里叶变换离子回旋质谱仪（FT-MS）分析。 对于20个样品中的每一个，在乙腈的2小时线性梯度上获得母体质量扫描。 将对照组和荷瘤组的质谱进行比对，并使用Rosetta ElucidatorTM软件定量各个准确的质量信号。使用两组之间具有统计学显著差异面积（P < 0.01）的同位素组来产生精确的m/z值，用于直接获取串联质谱以对肽进行测序并鉴定两组之间的蛋白质差异。在携带肿瘤的尿液中发现增加的蛋白质中（排除肝源性和公认的血液蛋白质，例如白蛋白、血红素结合蛋白、血红蛋白后）有甲氨蝶呤、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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.