South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Bellville, South Africa.
Department of Statistics and Population Studies, University of the Western Cape, Bellville, South Africa.
Pathol Oncol Res. 2021 May 3;27:622855. doi: 10.3389/pore.2021.622855. eCollection 2021.
To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age ( < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis ( > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.
为了阐明癌症发病机制及其分子水平的机制，需要收集和表征大量个体患者的组织队列。由于大多数病理研究所通常通过标准化的福尔马林固定和石蜡包埋方法来保存活检组织，因此这些存档的 FFPE 组织是包含患者元数据（如病史和治疗）的重要病理学材料的集合。由于福尔马林诱导的修饰，FFPE 块可以在环境条件下储存数十年，同时保持细胞形态。然而，在资源有限的发展中国家的机构中，长期储存对可提取蛋白质数量/质量的影响尚未得到研究。此外，对于来自不同年代 FFPE 组织块的无标记 LC-MS/MS 分析，实现准确和可重复结果所需的最佳样品制备技术仍然不清楚。本研究调查了 1 年、5 年和 10 年人类结直肠癌切除组织的蛋白质提取效率，并评估了三种不同的无凝胶蛋白质纯化方法用于无标记 LC-MS/MS 分析。每个实验组选择 n = 17 名患者的样本量（实验功率 = 0.7 和 = 0.05，产生 70%置信水平）。根据蛋白质提取浓度、肽/蛋白质鉴定、方法重现性和效率、样品蛋白质组完整性（由于储存时间）以及根据生物过程、细胞成分和理化性质的蛋白质/肽分布来评估数据。数据可在 ProteomeXchange 中使用标识符 PXD017198 进行访问。结果表明，提取的蛋白质量与块龄显著相关（ < 0.0001），较旧的块提取的蛋白质量少于较新的块。去污剂去除板是最有效的和总体上可重复的蛋白质纯化方法，就肽和蛋白质鉴定的数量而言，其次是 MagReSyn SP3/HILIC 方法（具有珠上酶消化），最后是丙酮沉淀和甲酸再溶解方法。总体而言，结果表明，FFPE 组织的长期储存（通过蛋氨酸氧化测量）不会严重干扰回顾性蛋白质组分析（ > 0.1）。块龄主要影响初始蛋白质提取产率，不会广泛影响后续无标记 LC-MS/MS 分析结果。
