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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们使用了一种无标记的定量纳米ESI-HPLC质谱学方法，对对照组大鼠和4-羟基丁基(丁基)亚硝胺诱发的膀胱肿瘤大鼠的尿液进行了比较蛋白质组学分析。在本研究中，对照动物(n=10)和可触及的膀胱肿瘤患者(n=10)的尿蛋白经还原和烷基化后，依次用内切酶Lys C和胰酶消化。采用纳米级反相高效液相色谱分离，纳米喷雾电离，混合线性四极傅立叶变换离子回旋质谱仪(FT-MS)分析。对20个样品中的每一个样品在乙腈的2小时线性梯度上进行母体质量扫描。用Rosetta ElucidatorTM软件对来自对照组和荷瘤组的质谱图进行比对，并对单个准确的质量信号进行量化。利用两组间有统计学意义差异的同位素组(P&lt；0.01)产生准确的m/z值，用于直接获取串联质谱图，以对多肽进行测序并鉴定两组之间的蛋白质差异。在肿瘤患者尿液中发现的升高的蛋白中(剔除肝源性和公认的血液蛋白，如白蛋白、血粘蛋白、血红蛋白)包括梅普林、E-钙粘素、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. 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.