Characterization of the membrane proteome and N-glycoproteome in BV-2 mouse microglia by liquid chromatography-tandem mass spectrometry.

ABSTRACT

BACKGROUND: Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited. RESULTS: To generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses. CONCLUSIONS: Herein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases.