Abstract
BackgroundMicroglial 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.ResultsTo 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.ConclusionsHerein, 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.
Highlights
Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes
Overall experimental workflow for membrane proteome and N-glycoproteome To achieve maximum coverage of the membrane proteome and N-glycoproteome in a reasonable time, we performed a novel proteomic analysis using a combination of crude membrane (CM) fractionation and protein digestion strategies without extensive peptide fractionation (Figure 1A)
LysC and trypsin were added to the sample from CM methods 1 and 2, yielding 2 digests
Summary
Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. 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. In response to pathological events, such as immunological stimuli, neuronal injury, and tissue Due to their function in immune and inflammation responses in the brain, microglia are recently considered central mediators in various neurological diseases, such as HIV-1-associated dementia, Alzheimer disease (AD), Parkinson disease (PD), tumors, brain and spinal cord trauma, stroke, and autoimmune CNS disease [6,7]. The molecular and cellular interactions between these proteins and their modification enable the cells to sense micro environmental variations and activate various mechanisms, including signaling pathways and transcriptional regulation of specific genes
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