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

对大鼠膀胱癌模型中的尿液进行有针对性的、无标记的蛋白质组学分析

• 批准号: 8168722
• 负责人: MING YOU
• 金额: $ 0.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168722
• 关键词: Acetonitriles; Albumins; Alkylation; Area; Binding Proteins; Bladder Neoplasm; Blood Proteins; CD44 Antigens; Cancer Model; Computer Retrieval of Information on Scientific Projects Database; Computer software; Control Animal; Cyclotrons; E-Cadherin; Fourier Transform; Funding; Grant; Hemoglobin; Hemopexin; Hepatic; High Pressure Liquid Chromatography; Hybrids; Individual; Institution; Ions; Isotopes; Label; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Meprin; Methods; Nitrosamines; Palpable; Parents; Peptides; Phase; Proteins; Proteomics; Rattus; Research; Research Personnel; Resources; Retinoblastoma; Sampling; Scanning; Signal Transduction; Source; Trypsin; United States National Institutes of Health; Urine; comparative; ionization; mass spectrometer; peptidyl-Lys metalloendopeptidase; protein aminoacid sequence; tumor; urinary

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们已经使用了无标记的定量纳米 - 纳米 - HPLC质谱法对对照大鼠和带有4-羟基丁基（丁基）硝基胺诱导的膀胱肿瘤的对照大鼠和大鼠进行尿液进行比较蛋白质组学分析。在这项研究中，对对照动物（n = 10）的尿蛋白以及可触及的膀胱肿瘤（n = 10）的尿蛋白被依次用内蛋白酶Lys C和还原和烷基化后的内蛋白酶Lys C和胰蛋白酶消化。用纳米流相反的相HPLC分离肽，并用纳米喷雾电离将肽离子化，并用杂交线性四极型傅立叶傅立叶转化离子转基环体质谱仪（FT-MS）进行分析。 对于20个样品中的每一个，在2小时的乙腈梯度上获得了母体质量扫描。 对照组对照组和承重肿瘤组的质谱进行对齐，并使用Rosetta Elucidatortm软件对单个精确的质量信号进行了定量。两组之间具有统计学意义不同区域（p <0.01）的同位素组用于产生准确的M/Z值，以定向串联质谱，以对肽进行序列并识别两组之间的蛋白质差异。在发现尿液中发现的蛋白质中（排除肝衍生和良好识别的血蛋白之后，例如白蛋白，血压素，血红蛋白）是E-钙粘蛋白，透明质酸受体，Glycam，Glycam，Glycam，视网膜细胞瘤结合蛋白。