{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T11:24:25Z","timestamp":1777634665073,"version":"3.51.4"},"reference-count":58,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T00:00:00Z","timestamp":1771977600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T00:00:00Z","timestamp":1771977600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteomics"],"published-print":{"date-parts":[[2026,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:label\/>\n                    <jats:p>Single\u2010cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single\u2010cell 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\u2010cell studies of microglia have focused on RNA\u2010sequencing 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\u2010assisted isolation, cryopreservation, and nanodroplet\u2010based processing for single\u2010cell mass spectrometry proteomics analysis of the postmortem human brain cortex\u2010derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof\u2010of\u2010principle, we applied single\u2010cell 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 that mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b\u2010c1 (Complex III), cytochrome c oxidase (Complex IV), F1\u2010ATPase (Complex V), and Na+\/K+\u2010ATPase 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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Significance of the Study<\/jats:title>\n                    <jats:p>Microglia are a key brain cell type that may contribute to pathogenesis in neurodegenerative disease. Transcriptomic profiling of microglia from the central nervous system of humans and animal models has identified several subtypes of microglia, and complementary proteomic profiling of microglia is likely to provide functionally and therapeutically relevant targets. Single\u2010cell proteomics studies of human\u2010derived microglia are lacking. This work describes a label\u2010free, single\u2010cell proteomics approach for microglia isolated by fluorescence\u2010activated cell sorting from a human donor that yields comparable numbers of identifications in comparison to prior single\u2010cell RNA sequencing studies of microglia. This approach holds promise for enabling large\u2010scale proteomics\u2010based subtyping of microglia and studying their roles in neurodegenerative diseases.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/pmic.70112","type":"journal-article","created":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T21:17:39Z","timestamp":1773004659000},"page":"59-68","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single\u2010Cell Nanodroplet Processing Proteomics Pipeline for Analysis of Human\u2010Derived Microglia"],"prefix":"10.1002","volume":"26","author":[{"given":"Ashley N.","family":"Ives","sequence":"first","affiliation":[{"name":"Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory  Richland Washington USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James M.","family":"Fulcher","sequence":"additional","affiliation":[{"name":"Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory  Richland Washington USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alex R.","family":"Bautista","sequence":"additional","affiliation":[{"name":"Center For Translational and Computational Neuroimmunology Department of Neurology &amp; Taub Institute for Research on Alzheimer's disease and the Aging Brain Columbia University Medical Center  New York USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reta Birhanu","family":"Kitata","sequence":"additional","affiliation":[{"name":"Biological Sciences Division Pacific Northwest National Laboratory  Richland Washington USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah M.","family":"Williams","sequence":"additional","affiliation":[{"name":"Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory  Richland Washington USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David A.","family":"Bennett","sequence":"additional","affiliation":[{"name":"Department of Neurological Sciences Rush University Medical Center  Chicago Illinois USA"},{"name":"Rush Alzheimer's Disease Center Rush University Medical Center  Chicago Illinois USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip L.","family":"De Jager","sequence":"additional","affiliation":[{"name":"Center For Translational and Computational Neuroimmunology Department of Neurology &amp; Taub Institute for Research on Alzheimer's disease and the Aging Brain Columbia University Medical Center  New York USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vladislav A.","family":"Petyuk","sequence":"additional","affiliation":[{"name":"Biological Sciences Division Pacific Northwest National Laboratory  Richland Washington 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