Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues.
Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues.
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DOI：
10.1021/pr7006768
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发表时间：
2008-02
影响因子：
4.4
通讯作者：
Haifeng Xu;Li Yang;Weijie Wang;S. Shi;Cheng Liu;Y. Liu;X. Fang;C. Taylor;Cheng S. Lee;B. Balgley
中科院分区：
文献类型：
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作者：
Haifeng Xu;Li Yang;Weijie Wang;S. Shi;Cheng Liu;Y. Liu;X. Fang;C. Taylor;Cheng S. Lee;B. Balgley
福尔马林固定和石蜡包埋的组织占存档组织的绝大多数。通过基于猎枪的蛋白质组学分析获得这样的组织标本可能为前瞻性和回溯性的翻译研究开辟新的途径。在这项研究中，我们评估固定时间对抗原回收的影响，目的是为了猎枪蛋白质组学。我们首次展示了基于毛细管等速电泳法(CITP)的蛋白质组学平台对从FFPE组织中回收的蛋白质进行鸟枪式蛋白质组学分析的能力。与我们之前使用毛细管等电聚焦的研究相比，基于CITP的分析更稳健，并增加了蛋白质组的覆盖率。在这种情况下，来自三个FFPE肝组织的结果总共产生了4098个不同的Swiss-Prot鉴定，错误发现率为1%。为了判断这些指认的准确性，对17种常见的检测蛋白质进行了免疫组织化学检测。这些蛋白质跨越了广泛的蛋白质丰度，通过光谱计数从相对定量中推断出来。在该小组中，有4种蛋白质由单一的多肽HIT鉴定，包括三个分化(CD)标记：CD74、CD117和CD45。由于单肽撞击通常被认为是怀疑的，值得注意的是，IHC检测的所有蛋白质都呈阳性。
Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.