Proteome, Phosphoproteome, and N-Glycoproteome Are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry
Proteome, Phosphoproteome, and N-Glycoproteome Are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry
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DOI：
10.1021/pr100234w
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发表时间：
2010-07-01
影响因子：
4.4
通讯作者：
Wisniewski, Jacek R.
中科院分区：
文献类型：
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作者：
Ostasiewicz, Pawel;Zielinska, Dorota F.;Wisniewski, Jacek R.
生物库中的组织样本通常是福尔马林固定和石蜡包埋的（FFPE），以这种形式保存数十年。直到最近才表明，FFPE组织中的蛋白质可以通过基于质谱的蛋白质组学来鉴定，但翻译后修饰的分析被认为是困难或不可能的。过滤器辅助样品制备（FASP）方法可以分析蛋白质样品溶解在高浓度的SDS，我们使用这个功能，开发一个简单的协议FFPE分析。结合简单的基于移液器尖端的肽分级，在24小时的测量时间内鉴定了约5000种小鼠肝脏蛋白质，与新鲜组织中的相同。FFPE-FASP程序的结果未显示由于储存时间、苏木精染色或激光捕获显微切割而导致的任何可辨别的变化。我们使用SILAC小鼠将新鲜组织与FFPE组织进行了比较，发现这些样品之间在蛋白质或肽水平上没有显著的定性或定量差异。将我们的FFPE-FASP方案应用于磷酸化和N-糖基化，精确定位了近5000个磷酸化位点和1500个N-糖基化位点。SILAC小鼠的FFPE组织分析显示，这些翻译后修饰被定量保留。因此，FFPE生物库材料可以通过定量蛋白质组学在蛋白质和翻译后修饰水平上进行分析。
Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteonnic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.