title: Macrophages Cell Surface Proteome Analysis for Identification of Markers and Therapeutic Targets
Creators: name: Cohen, Benjamin
Publisher: name: UNSPECIFIED
Year: 2024

description: Macrophages are the first line of defense against common pathogens and play an important role in the homeostatic maintenance of the body. Their activities and mode of action can be diverse depending on the origin of the cells and the signal that caused their activation (bacteria, dead cells, tumors, etc.). Differentiation of monocytes into Macrophages can be classified into 2 main groups: classically activated macrophages (M1 Macrophages), associated with microbicidal and anti-tumor activity, and alternatively activated macrophages (M2 Macrophages) which promote cell proliferation and tissue repair. There has been explosive growth in macrophage-targeted therapy during the past decade. With more than 600 clinical trials conducted for tumor-associated macrophages (TAMs) (S. Wang, Yang, Ma, Huang, et al., 2022), there is a growing appetite for discovery of new and innovative targets to detect and approach macrophages.
There has been no direct surfaceome comparison of the most commonly researched macrophage sources, mouse bone marrow and human monocytes from peripheral blood, across multiple macrophage phenotypes. 
In this study, macrophages from different sources were analysed by cell surface proteomic level for the first time, using the cell surface proteome analysis (Cell SPA) method. The method comprises four main steps: (i) an initial biotinylation to tag the membranal proteins; (ii) cell lysis; (iii) precipitation on streptavidin columns (affinity pull-down); and (iv) stringent washes to enrich the membrane fraction and remove proteins that were bound non-specifically. By the end of this process, only biotinylated cell-surface proteins are isolated, and they are then characterized and identified by using LC-MS/MS.
Herein, 965 cell surface proteins for mouse and 452 for human macrophages were identified and classified as M1, M2 or M0. RNAseq was performed to verify the proteomic results and they were found to be highly correlative. Also, flow cytometry analysis was performed to validate the proteomics results.
All the known and expected proteins for each sample are found in the analysed data, and act as a foundation, anchoring and verifying the presence and precision of the remaining proteins. Some of the proteins that were found can serve as a new marker, and some hold potential for therapeutic value.
This work also provides information of differential cell surface proteins expression between human and mouse macrophages. It is widely known that proteins such as EMR1 (F4/80) are expressed only on mouse macrophages. This work shows that SLAMF1 protein is found solely in M1 macrophages in mice, and that P2RY11 protein is found to be exclusively expressed on human M2 macrophages.
Hence, information on macrophages from different origin should be carefully observed and analysed. Presence and levels of proteins expression can differ significantly between different origins. These differences can lead to misunderstandings and misinterpretations in further experiments particularly in the context of clinical experiments. 
Also, this present study demonstrates the potential of cell surface proteome analysis to explore the biology of cells and the potential to treat human diseases by revealing new targets.