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Single-cell western blotting (scWB) provides quantitative, molecular-mass-resolved protein measurements but relies on Poisson-limited gravity settling, requiring approximately 10\u2076 starting cells while constraining single-cell microwell occupancy to a theoretical maximum of 36.8%. Here, we introduce AutoBlot, an image-guided piezoelectric dispensing approach that deterministically loads individual cells into predefined scWB microwells. AutoBlot achieved approximately 98% single-cell occupancy among targeted microwells while operating from as few as 10,000 starting cells. Across the tested cell concentrations, measured occupancy exceeded Poisson predictions by 31- to 904-fold, and the same dispensing strategy enabled deterministic cell-bead co-loading. We applied AutoBlot to 1,449 cells from seven patient-derived breast organoids generated from histologically normal mammary tissue. Measurements of five lineage-associated proteins revealed donor-to-donor variation, differences associated with germline BRCA1 and menopausal status, and discordance between surface-marker- and cytokeratin-based lineage assignments. AutoBlot also resolved ER-immunoreactive species at approximately 66 and 46 kDa in individual PDO cells. These results establish deterministic cell loading as a strategy for extending molecular-mass-resolved single-cell protein analysis to heterogeneous, cell-limited specimens.<\/jats:p>","DOI":"10.64898\/2026.06.22.733890","type":"posted-content","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T16:25:14Z","timestamp":1782231914000},"source":"Crossref","is-referenced-by-count":2,"title":["AutoBlot: deterministic single-cell western blotting reveals proteomic diversity in rare cell populations"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8909-0847","authenticated-orcid":false,"given":"Cyril","family":"Deroy","sequence":"first","affiliation":[{"name":"Department of Bioengineering, University of California, Berkeley, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4944-7439","authenticated-orcid":false,"given":"Md Nazibul","family":"Islam","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of California, Berkeley, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1644-3984","authenticated-orcid":false,"given":"Nadine","family":"Goldhammer","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Francisco, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorraine","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Francisco, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1314-6976","authenticated-orcid":false,"given":"Jennifer M.","family":"Rosenbluth","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Francisco, USA"},{"name":"Chan Zuckerberg Biohub, San Francisco, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6906-2985","authenticated-orcid":false,"given":"Amy E.","family":"Herr","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of California, Berkeley, USA"},{"name":"Chan Zuckerberg Biohub, San Francisco, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026090709550980000_2026.06.22.733890v2.1","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/s41570-020-0162-7","article-title":"Single-cell analysis targeting the proteome","volume":"4","year":"2020","journal-title":"Nat. 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