Amarnani Abhimanyu, Capri Joseph R, Souda Puneet, Elashoff David A, Lopez Ivan A, Whitelegge Julian P, Singh Ram R
Department of Head and Neck Surgery, UCLA, Los Angeles, CA 90095, USA.
Department of Medicine/Rheumatology, UCLA, Los Angeles, CA 90095, USA.
J Proteomics Bioinform. 2019;12(7):104-112. doi: 10.35248/0974-276X.12.19.503. Epub 2019 Oct 3.
Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases.
Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS.
We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, 'systemic lupus erythematosus', 'interferon-α', 'TGF-β', and 'extracellular matrix', were reproducibly quantified between FFPE and FFTs. However, 12%-29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including 'inflammatory response' and 'innate immune system' were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys.
This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.
由于难以获取用于全球数据驱动方法的新鲜冷冻组织（FFT），人类疾病发病机制的研究受到了阻碍。作为替代方案，病理库中福尔马林固定石蜡包埋（FFPE）组织很容易获得。然而，使用福尔马林固定会导致炎症过程中出现的蛋白质丢失，从而引入固有的样本偏差。为了解决这个问题，我们比较了FF和FFPE组织蛋白质组学，以确定FFPE组织是否能有效地用于炎症性疾病研究。
将狼疮性肾炎小鼠的相邻肾脏切片制成FFPE或FFT样本。它们的组织裂解物通过蛋白质组学流程一起进行分析，该流程包括过滤辅助样本制备、溶液内二甲基同位素标记、StageTip分级分离以及通过Orbitrap XL质谱仪进行的纳升液相色谱串联质谱分析。
我们报告称，在小鼠狼疮性肾炎肾脏中，相邻FFPE和FFT样本之间的蛋白质鉴定一致性超过97%。具体而言，代表“系统性红斑狼疮”“干扰素-α”“转化生长因子-β”和“细胞外基质”等通路的蛋白质在FFPE和FFT样本之间能够被重复定量。然而，与FFT样本相比，FFPE样本中有12% - 29%的蛋白质定量存在差异，但这些差异在各个实验中是一致的。特别是，“炎症反应”和“固有免疫系统”等通路中的某些蛋白质在FFPE样本中的定量低于FFT样本。在一项针对人类FFPE组织的初步研究中，我们鉴定出了狼疮性肾炎肾脏活检组织与对照肾脏中与发病机制相关的蛋白质。
这是首次使用FFPE组织进行狼疮性肾炎肾脏蛋白质组学研究的报告。我们得出结论，存档的FFPE组织可可靠地用于炎症性疾病的蛋白质组学分析，但需要注意的是，与免疫和炎症相关的某些蛋白质在FFPE样本中的定量可能低于FFT样本中的定量。
