Single-cell nanodroplet processing proteomics pipeline for analysis of human-derived microglia

Single-cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single-cell RNA sequencing has identified several transcriptionally distinct populations of microglia, which are resident immune cells of the central nervous system (CNS) that are responsive to CNS injury, infection, and neurodegeneration. To date, single cell studies of microglia have focused on RNA-sequencing or cytometry by time of flight (CyTOF) which provide indirect readouts of protein abundance or quantification of a limited number of targets. Herein, we present a workflow based on FACS-assisted isolation, cryopreservation and nanodroplet-based processing for single-cell mass spectrometry proteomics analysis of the postmortem human brain cortex-derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof-of-principle we applied single-cell proteomics for exploring the heterogeneity of brain microglia at the cellular level. This pilot proteomics data partially recapitulates the prior microglia subtypes. Specifically, we determined mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b-c1 (Complex III), cytochrome c oxidase (Complex IV), F1-ATPase (Complex V), and Na+/K+-ATPase complex drive variation across microglia. This pipeline offers the potential for identifying functionally and analytically relevant protein targets for microglia in Alzheimer’s disease and other neurological disorders.