Haines Moe, Thorup John R, Gohsman Simone, Ctortecka Claudia, Newton Chelsea, Rohrer Dan C, Hostetter Galen, Mani D R, Gillette Michael A, Satpathy Shankha, Carr Steven A
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Van Andel Research Institute, Grand Rapids, MI, USA.
bioRxiv. 2024 Dec 7:2024.11.17.624038. doi: 10.1101/2024.11.17.624038.
Formalin-fixed, paraffin-embedded (FFPE) patient tissues are a valuable resource for proteomic studies with the potential to associate the derived molecular insights with clinical annotations and outcomes. Here we present an optimized, partially automated workflow for FFPE proteomics combining pathology-guided macro-dissection, Adaptive Focused Acoustics (AFA) sonication for lysis and decrosslinking, S-Trap digestion to peptides, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using Orbitrap, Astral or timsTOF HT instrumentation. The workflow enables analysis of up to 96 dissected FFPE tissue samples or 10 μm scrolls, identifying 8,000-10,000 unique proteins per sample with median CVs <20%. Key optimizations include improved tissue lysis strategies, protein quantification for normalization, and peptide cleanup prior to LC-MS/MS analysis. Application to lung adenocarcinoma (LUAD) FFPE blocks confirms the platform's effectiveness in processing complex, clinically relevant samples, achieving deep proteome coverage and quantitative robustness comparable to TMT-based methods. Using the newly released Orbitrap Astral with short, 24-minute gradients, the workflow identifies up to ~10,000 unique proteins and ~11,000 localized phosphosites in LUAD FFPE tissue. This high-throughput, scalable workflow advances biomarker discovery and proteomic research in archival tissue samples.
福尔马林固定、石蜡包埋（FFPE）的患者组织是蛋白质组学研究的宝贵资源，有可能将获得的分子见解与临床注释和结果联系起来。在这里，我们展示了一种优化的、部分自动化的FFPE蛋白质组学工作流程，该流程结合了病理学指导的宏观解剖、用于裂解和去交联的自适应聚焦声学（AFA）超声处理、S-Trap酶解成肽以及使用Orbitrap、Astral或timsTOF HT仪器的液相色谱-串联质谱（LC-MS/MS）分析。该工作流程能够分析多达96个解剖的FFPE组织样本或10μm切片，每个样本可鉴定8000-10000种独特蛋白质，中位数变异系数<20%。关键的优化包括改进的组织裂解策略、用于归一化的蛋白质定量以及LC-MS/MS分析前的肽段净化。应用于肺腺癌（LUAD）FFPE样本块证实了该平台在处理复杂的、临床相关样本方面的有效性，实现了与基于TMT的方法相当的深度蛋白质组覆盖和定量稳健性。使用新发布的具有24分钟短梯度的Orbitrap Astral，该工作流程在LUAD FFPE组织中鉴定出多达约10000种独特蛋白质和约11000个定位的磷酸化位点。这种高通量、可扩展的工作流程推动了存档组织样本中的生物标志物发现和蛋白质组学研究。
