浸润肿瘤微环境 (TME) 的免疫细胞在塑造癌症发展、影响临床结果和治疗反应方面发挥着至关重要的作用。然而，在临床标本中获得肿瘤浸润免疫的全面蛋白质组学快照常常受到样本量小和 TME 中免疫浸润细胞比例低的阻碍。为了能够对微型组织进行深入且高度敏感的分析，我们建立了一种免疫细胞富集库辅助策略，用于数据独立的采集质谱（DIA-MS）。首先，从小鼠肠系膜淋巴结（MLN）中分选的CD8、CD4 T淋巴细胞、B淋巴细胞、自然杀伤细胞、树突状细胞和巨噬细胞建立了6个免疫细胞亚型特异性谱库，涵盖7,815个具有表面标记和免疫功能的蛋白组。富含细胞的蛋白质。使用单次 DIA 对来自小鼠结直肠癌 (CRC) 模型肿瘤的 1 μg 组织蛋白进行微量免疫蛋白质组分析的可行性得到了证明；与直接DIA分析（6,978个蛋白质）相比，免疫细胞富集文库增加了量化7,419个蛋白质的覆盖范围。这一增强功能能够绘制 841 个免疫功能相关蛋白的图谱，并独家鉴定许多低丰度的免疫蛋白，例如 CD1D1 和 CD244，证明了免疫景观分析的高灵敏度。该方法用于表征 CRC 模型中的 MLN，旨在阐明它们参与 TME 内癌症发展的潜在机制。即使肿瘤中免疫细胞浸润比例较低（0.25-3%），我们的结果也阐明了适应性免疫信号传导途径（C 型凝集素受体信号传导、趋化因子信号传导等）、T 细胞受体信号传导的下调和 Th1/Th2/Th17 细胞分化，表明 CRC 模型的 MLN 中存在免疫抑制状态。使用免疫细胞富集文库的 DIA 方法展示了深度覆盖和高灵敏度，有助于阐明微量样品的免疫蛋白质组景观。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, 6 immune cell subtype-specific spectral libraries were established from sorted CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7,815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7,419 proteins compared to directDIA analysis (6,978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundant immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was employed to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25-3%) in the tumor, our results illuminate down-regulation in the adaptive immune signaling pathways (C-type lectin receptor signaling, chemokine signaling, etc.), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC models. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.