Proteome, Phosphoproteome, and N-Glycoproteome Are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry

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 proteomic 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.

生物样本库（biobanks）中的组织样本通常采用福尔马林固定石蜡包埋（formalin-fixed and paraffin-embedded，FFPE）的方式保存，此类样本可留存数十年之久。直到近年才有研究证实，基于质谱的蛋白质组学（mass spectrometry-based proteomics）可对FFPE组织中的蛋白质进行鉴定，但学界普遍认为对其翻译后修饰（post-translational modifications）的分析难度极高，甚至无法实现。滤助样品制备（filter aided sample preparation，FASP）法可分析以高浓度十二烷基硫酸钠（sodium dodecyl sulfate，SDS）溶解的蛋白质组学样本，我们依托这一特性开发了一套适用于FFPE样本分析的简易实验方案。结合基于移液吸头的简易肽段分级分离技术，我们在24小时的检测时长内成功鉴定出约5000个小鼠肝脏蛋白质，这一结果与新鲜组织样本的检测水平相当。FFPE-FASP流程的检测结果未显示出因存储时长、苏木精染色（hematoxylin staining）或激光捕获显微切割（laser capture microdissection）所导致的可辨识差异。我们采用稳定同位素标记氨基酸细胞培养（stable isotope labeling with amino acids in cell culture，SILAC）小鼠模型对比了新鲜组织与FFPE组织样本，结果表明二者在蛋白质及肽段层面均无显著的定性或定量差异。将该FFPE-FASP方案应用于磷酸化（phosphorylation）与N-糖基化（N-glycosylation）分析，我们分别定位到近5000个磷酸化位点与1500个N-糖基化位点。对SILAC小鼠的FFPE组织进行分析后发现，这些翻译后修饰在定量层面得到了完好保留。综上，生物样本库中的FFPE组织样本可通过定量蛋白质组学技术，在蛋白质及翻译后修饰层面实现全面分析。