Allsup Benjamin L, Gharpure Supriya, Bryson Bryan D
Department of Biological Engineering, MIT, Cambridge, USA.
Ragon Institute of Mass General, Harvard, and MIT, Cambridge, USA.
bioRxiv. 2024 Sep 8:2024.09.04.611277. doi: 10.1101/2024.09.04.611277.
Proteomic analyses of the phagosome has significantly improved our understanding of the proteins which contribute to critical phagosome functions such as apoptotic cell clearance and microbial killing. However, previous methods of isolating phagosomes for proteomic analysis have relied on cell fractionation with some intrinsic limitations. Here, we present an alternative and modular proximity-labeling based strategy for mass spectrometry proteomic analysis of the phagosome lumen, termed PhagoID. We optimize proximity labeling in the phagosome and apply PhagoID to immortalized murine macrophages as well as primary human macrophages. Analysis of proteins detected by PhagoID in murine macrophages demonstrate that PhagoID corroborates previous proteomic studies, but also nominates novel proteins with unexpected residence at the phagosome for further study. A direct comparison between the proteins detected by PhagoID between mouse and human macrophages further reveals that human macrophage phagosomes have an increased abundance of proteins involved in the oxidative burst and antigen presentation. Our study develops and benchmarks a new approach to measure the protein composition of the phagosome and validates a subset of these findings, ultimately using PhagoID to grant further insight into the core constituent proteins and species differences at the phagosome lumen.
对吞噬体的蛋白质组学分析显著增进了我们对有助于凋亡细胞清除和微生物杀伤等关键吞噬体功能的蛋白质的理解。然而，以往用于蛋白质组学分析的吞噬体分离方法依赖细胞分级分离，存在一些固有局限性。在此，我们提出一种基于邻近标记的替代模块化策略，用于对吞噬体腔进行质谱蛋白质组学分析，称为PhagoID。我们优化了吞噬体中的邻近标记，并将PhagoID应用于永生化小鼠巨噬细胞以及原代人巨噬细胞。对PhagoID在小鼠巨噬细胞中检测到的蛋白质进行分析表明，PhagoID证实了以往的蛋白质组学研究，但也鉴定出在吞噬体中意外存在的新蛋白质以供进一步研究。对小鼠和人巨噬细胞中PhagoID检测到的蛋白质进行直接比较，进一步揭示人巨噬细胞吞噬体中参与氧化爆发和抗原呈递的蛋白质丰度增加。我们的研究开发并评估了一种测量吞噬体蛋白质组成的新方法，并验证了其中一部分发现，最终利用PhagoID进一步深入了解吞噬体腔中的核心组成蛋白质和物种差异。
