Shah Punit, Zhang Bai, Choi Caitlin, Yang Shuang, Zhou Jianying, Harlan Robert, Tian Yuan, Zhang Zhen, Chan Daniel W, Zhang Hui
Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
Anal Biochem. 2015 Jan 15;469:27-33. doi: 10.1016/j.ab.2014.09.017. Epub 2014 Oct 2.
Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.
蛋白质组学分析对于表征组织以获得生物学和病理学见解非常重要，这可能会导致识别与疾病相关的蛋白质用于疾病诊断或靶向治疗。然而，为了保持组织形态以便进行组织学评估，组织通常被包埋在最佳切割温度介质（OCT）中，或者进行福尔马林固定和石蜡包埋（FFPE）。尽管已经使用先进的质谱（MS）技术对FFPE组织进行了多项组织蛋白质组学分析，但由于OCT对MS分析的干扰，对OCT包埋组织进行高通量蛋白质组学分析一直很困难。此外，组织中存在的除蛋白质之外的分子进一步使组织蛋白质组学分析复杂化。在此，我们报告了一种使用蛋白质化学固定用于肽提取（CIPPE）的方法的开发。在这种方法中，蛋白质被化学固定在固体支持物上；通过对结合在固体支持物上的蛋白质进行大量洗涤，去除组织和OCT包埋的干扰。然后通过蛋白水解从固相中释放肽，从而能够进行MS分析。该方法首先通过消除来自标准蛋白质人血清白蛋白的OCT干扰进行验证，其中由OCT污染产生的所有独特峰都被消除。最后，该方法应用于使用iTRAQ（相对和绝对定量的等压标签）标记和二维液相色谱串联质谱对冷冻和OCT包埋组织进行蛋白质组学分析。数据显示从3996个蛋白质组中可重复提取和定量10284种蛋白质，并且OCT包埋对储存组织样品的整体蛋白质组分析的影响最小。
