{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T17:29:58Z","timestamp":1793294998775,"version":"4.5.0"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"38","license":[{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000997","name":"Arnold and Mabel Beckman Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000997","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000049","name":"National Institute on Aging","doi-asserted-by":"publisher","award":["R01AG088147"],"award-info":[{"award-number":["R01AG088147"]}],"id":[{"id":"10.13039\/100000049","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Direct proteome analysis of identified neurons in intact brain tissue remains limited by the difficulty of recovering intracellular material while preserving native tissue context and avoiding physical extraction of the intact soma. Here, we establish aspiration patch proteomics as a mass spectrometry (MS)-compatible microsampling strategy for proteome-level analysis of somal cytoplasmic aspirates from fluorescently identified neurons in acute mouse brain slices. The workflow combines patch-pipet aspiration, volatile internal-solution chemistry, minimal-loss bottom-up proteomics, and high-sensitivity capillary electrophoresis (CE)\u2013electrospray ionization (ESI)\u2013MS on a timsTOF platform. Optimization of the patch internal solution revealed a strong trade-off between electrophysiological compatibility and proteomic depth, identifying ammonium bicarbonate as an enabling volatile electrolyte for downstream CE\u2013ESI\u2013MS analysis. Applied to dopaminergic, parvalbumin, and serotonergic neurons, the method identified hundreds to &amp;gt;1000 proteins from optimized single-neuron somal cytoplasmic aspirate measurements while analyzing only \u223c0.4% of the processed digest per CE\u2013ESI\u2013MS run. Across neuronal classes, 1894 protein entries were identified and 1703 were quantified, yielding reproducible label-free profiles sufficient to separate biological replicates by neuronal phenotype and distinguish neuronal classes from protein expression alone. These results show that aspiration-based recovery of somal cytoplasm yields sufficient proteomic information for subtype-level single-neuron proteotyping while maintaining intact-tissue targeting and avoiding cell dissociation or physical extraction of the intact soma. Aspiration patch proteomics therefore provides a chemically compatible sampling front end for MS-based analysis of identified neurons in native brain tissue.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c03049","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T16:01:49Z","timestamp":1786118509000},"page":"28088-28096","source":"Crossref","is-referenced-by-count":1,"title":["Aspiration\nPatch Proteomics Enables CE\u2013ESI\u2013MS\nProteotyping of Identified Single Neurons in Intact Brain Tissue"],"prefix":"10.1021","volume":"98","author":[{"given":"Cole C.","family":"Johnson","sequence":"first","affiliation":[{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sam B.","family":"Choi","sequence":"additional","affiliation":[{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan A.","family":"Zegers-Delgado","sequence":"additional","affiliation":[{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandre","family":"Kisner","sequence":"additional","affiliation":[{"name":"The George Washington University , , , ,","place":["Washington, District of Columbia, United States, 20037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo C.","family":"Araneda","sequence":"additional","affiliation":[{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]},{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abigail M.","family":"Polter","sequence":"additional","affiliation":[{"name":"The George Washington University , , , ,","place":["Washington, District of Columbia, United States, 20037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4704-4997","authenticated-orcid":true,"given":"Peter","family":"Nemes","sequence":"additional","affiliation":[{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]},{"name":"University of Maryland , , , ,","place":["College Park, Maryland, United States, 20742"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"issue":"1","key":"2026092909103398400_cit1","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1038\/nn.3881","article-title":"Unbiased classification of sensory neuron types by\nlarge-scale single-cell RNA sequencing","volume":"18","author":"Usoskin","year":"2015","journal-title":"Nat.\nNeurosci."},{"issue":"7697","key":"2026092909103398400_cit2","doi-asserted-by":"publisher","first-page":"524","DOI":"10.1038\/nature25980","article-title":"A single-cell RNA-seq survey of the\ndevelopmental landscape of the\nhuman prefrontal cortex","volume":"555","author":"Zhong","year":"2018","journal-title":"Nature"},{"issue":"10","key":"2026092909103398400_cit3","doi-asserted-by":"publisher","first-page":"2689","DOI":"10.1016\/j.celrep.2018.11.034","article-title":"Single-cell\nRNA-Seq of mouse olfactory\nbulb reveals cellular heterogeneity and activity-dependent molecular\ncensus of adult-born neurons","volume":"25","author":"Tepe","year":"2018","journal-title":"Cell Rep."},{"issue":"2","key":"2026092909103398400_cit4","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1038\/nn.4216","article-title":"Adult mouse cortical\ncell taxonomy\nrevealed by single cell transcriptomics","volume":"19","author":"Tasic","year":"2016","journal-title":"Nat.\nNeurosci."},{"issue":"13","key":"2026092909103398400_cit5","doi-asserted-by":"publisher","first-page":"3227","DOI":"10.1016\/j.celrep.2017.03.004","article-title":"Single-cell RNA-Seq reveals hypothalamic\ncell diversity","volume":"18","author":"Chen","year":"2017","journal-title":"Cell Rep."},{"key":"2026092909103398400_cit6","doi-asserted-by":"publisher","first-page":"No. 1007755","DOI":"10.3389\/fncir.2023.1007755","article-title":"Recent advances\nin understanding neuronal diversity\nand neural circuit complexity across different brain regions using\nsingle-cell sequencing","volume":"17","author":"Xing","year":"2023","journal-title":"Front. 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Neurosci."},{"issue":"9","key":"2026092909103398400_cit37","doi-asserted-by":"publisher","first-page":"786","DOI":"10.1038\/sj.mp.4001379","article-title":"Neuronal expression of GFAP in patients\nwith Alzheimer\npathology and identification of novel GFAP splice forms","volume":"8","author":"Hol","year":"2003","journal-title":"Mol. Psychiatry"},{"issue":"6","key":"2026092909103398400_cit38","doi-asserted-by":"publisher","first-page":"2323","DOI":"10.1007\/s00414-021-02568-1","article-title":"GFAP positivity\nin neurons following traumatic brain injuries","volume":"135","author":"Zwirner","year":"2021","journal-title":"Int. J. Leg. 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