Clinical Proteomics Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
J Pathol. 2020 May;251(1):100-112. doi: 10.1002/path.5420.
Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
福尔马林固定和石蜡包埋（FFPE）是保存人类组织进行临床诊断的最常用方法，FFPE 档案是生物医学研究的宝贵资源。FFPE 材料中的蛋白质在几十年内都是稳定的，但通过基于质谱（MS）的蛋白质组学对其进行高效提取和简化分析一直具有挑战性。本文中，我们描述了一种基于 MS 的蛋白质组学工作流程，用于直接从组织病理学载玻片定量分析大型 FFPE 组织队列。我们证明了该工作流程在临床病理学标本和不同的样本量上具有广泛的适用性，包括通过激光微切割分离的低输入量的癌症组织。使用最先进的基于数据依赖采集（DDA）和数据独立采集（DIA）MS 工作流程，我们在 100 分钟的单次运行分析中始终能够定量分析大部分蛋白质组。在包含 100 多个样本的腺瘤队列中，总工作量不到一天。我们观察到，长期储存的样本（>15 年）中的蛋白质鉴定数量呈适度下降趋势，但聚类成不同的蛋白质组亚型与存档时间无关。我们的研究结果强调了使用无偏蛋白质组学对 FFPE 组织进行患者表型分析的巨大潜力，并证明了以稳健、及时和简化的方式分析大型组织队列的可行性。© 2020 作者。病理学杂志由 John Wiley & Sons Ltd 代表英国和爱尔兰病理学会出版。
