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Due to its high compatibility with mass spectrometry (MS), n-Dodecyl-d-maltoside (DDM) is commonly used for protein extraction from small tissue sections in spatially resolved proteomics workflows, improving low-input sample recovery without surfactant removal prior to MS analysis. However, extracting and solubilizing highly hydrophobic membrane proteins using mild, nondenaturing detergents remains challenging. Herein, we propose a formic acid (FA)-enhanced single-pot sample preparation (FAESP) method to improve protein solubility and extraction from tissue samples, enhancing membrane proteome identification coverage in laser capture microdissected tissue sections. Due to its high volatility, FA is rapidly removed by vacuum drying without cleaning steps. Combination with DDM ensures compatibility with subsequent digestion and LC-MS analysis. Compared with the FA-free DDM-based method, FAESP achieves a 38% increase in proteome coverage, with 40% of identified proteins annotated as membrane proteins. The FAESP workflow was applied to neuron type-specific proteome profiling in the medial habenula (MHb) of mouse brain, revealing regional specificity of synaptic proteins and solute carrier proteins in aging MHb subregions. These results demonstrate that FAESP provides a superior tool for discovering membrane protein candidates that distinguish neuronal types, facilitating biological and clinical applications.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c01074","type":"journal-article","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T17:40:39Z","timestamp":1784828439000},"page":"22017-22024","source":"Crossref","is-referenced-by-count":0,"title":["Formic Acid\nEnhanced Single-Pot Sample Preparation\nto Improve Identification Coverage of Membrane Proteome of Laser Capture\nMicrodissected Tissue Sections"],"prefix":"10.1021","volume":"98","author":[{"given":"Yingyun","family":"He","sequence":"first","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]},{"name":"University of Chinese Academy of Sciences , ,","place":["Beijing, China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyu","family":"He","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]},{"name":"Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences , , ,","place":["Guangzhou, China, 510530"]},{"name":"University of Chinese Academy of Sciences , ,","place":["Beijing, China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1280-309X","authenticated-orcid":true,"given":"Dan","family":"Liu","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinfang","family":"Xie","sequence":"additional","affiliation":[{"name":"Shenzhen-Hong Kong Institute of Brain Science , , ,","place":["Shenzhen, China, 518055"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahang","family":"Tang","sequence":"additional","affiliation":[{"name":"Shenzhen-Hong Kong Institute of Brain Science , , ,","place":["Shenzhen, China, 518055"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3540-8893","authenticated-orcid":true,"given":"Baofeng","family":"Zhao","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-2224-7038","authenticated-orcid":true,"given":"Jianhui","family":"Liu","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Liang","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin-an","family":"Liu","sequence":"additional","affiliation":[{"name":"Shenzhen-Hong Kong Institute of Brain Science , , ,","place":["Shenzhen, China, 518055"]},{"name":"University of Chinese Academy of Sciences , ,","place":["Beijing, China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2543-1547","authenticated-orcid":true,"given":"Lihua","family":"Zhang","sequence":"additional","affiliation":[{"name":"Dalian Institute of Chemical Physics, Chinese Academy of Sciences , , ,","place":["Dalian, China, 116023"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1893-7294","authenticated-orcid":true,"given":"Huiming","family":"Yuan","sequence":"additional","affiliation":[{"name":"Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences , , ,","place":["Guangzhou, China, 510530"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,23]]},"reference":[{"issue":"5","key":"2026080408074318500_cit1","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1038\/s41580-018-0094-y","article-title":"Spatial proteomics: a powerful discovery tool for cell\nbiology","volume":"20","author":"Lundberg","year":"2019","journal-title":"Nat. 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Biol."},{"issue":"1","key":"2026080408074318500_cit5","doi-asserted-by":"publisher","DOI":"10.1038\/srep39223","article-title":"Spatially-Resolved\nProteomics: Rapid Quantitative Analysis of Laser Capture Microdissected\nAlveolar Tissue Samples","volume":"6","author":"Clair","year":"2016","journal-title":"Sci. Rep."},{"issue":"4\u20135","key":"2026080408074318500_cit6","doi-asserted-by":"publisher","first-page":"403","DOI":"10.1002\/mas.21598","article-title":"Proteome analysis of tissues by mass spectrometry","volume":"38","author":"Dapic","year":"2019","journal-title":"Mass Spectrom. Rev."},{"issue":"3","key":"2026080408074318500_cit7","doi-asserted-by":"publisher","first-page":"1587","DOI":"10.1021\/pr5012679","article-title":"New mass-spectrometry-compatible\ndegradable surfactant for tissue proteomics","volume":"14","author":"Chang","year":"2015","journal-title":"J. Proteome Res."},{"issue":"4","key":"2026080408074318500_cit8","doi-asserted-by":"publisher","first-page":"2054","DOI":"10.1021\/ac504700t","article-title":"High-sensitivity N-glycoproteomic\nanalysis of mouse brain tissue by protein extraction with a mild detergent\nof N-dodecyl beta-D-maltoside","volume":"87","author":"Liu","year":"2015","journal-title":"Anal. Chem."},{"issue":"9","key":"2026080408074318500_cit9","doi-asserted-by":"publisher","first-page":"5879","DOI":"10.1021\/acs.analchem.8b00596","article-title":"Spatial-Resolution Cell\nType Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics\nTechnology","volume":"90","author":"Xu","year":"2018","journal-title":"Anal. 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Proteomics"},{"issue":"23","key":"2026080408074318500_cit15","doi-asserted-by":"publisher","first-page":"9622","DOI":"10.1021\/ac1023099","article-title":"Coupling formic\nacid assisted solubilization and online immobilized pepsin digestion\nwith strong cation exchange and microflow reversed-phase liquid chromatography\nwith electrospray ionization tandem mass spectrometry for integral\nmembrane proteome analysis","volume":"82","author":"Ma","year":"2010","journal-title":"Anal. Chem."},{"issue":"18","key":"2026080408074318500_cit16","doi-asserted-by":"publisher","first-page":"8507","DOI":"10.1021\/ac402076u","article-title":"Biphasic microreactor\nfor efficient membrane protein pretreatment with a combination of\nformic acid assisted solubilization, on-column pH adjustment, reduction,\nalkylation, and tryptic digestion","volume":"85","author":"Zhao","year":"2013","journal-title":"Anal. Chem."},{"issue":"1","key":"2026080408074318500_cit17","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1074\/mcp.TIR119.001616","article-title":"Sample Preparation\nby Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and\nDetergent-free Protocol for Proteomics Based on Acid Extraction","volume":"19","author":"Doellinger","year":"2020","journal-title":"Mol. Cell. Proteomics"},{"issue":"6","key":"2026080408074318500_cit18","doi-asserted-by":"publisher","first-page":"3291","DOI":"10.1021\/acs.analchem.2c04264","article-title":"ASAP horizontal\nline Automated Sonication-Free Acid-Assisted Proteomes horizontal\nline from Cells and FFPE Tissues","volume":"95","author":"Barnabas","year":"2023","journal-title":"Anal. 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Neurobiol."},{"issue":"12","key":"2026080408074318500_cit24","doi-asserted-by":"publisher","DOI":"10.3390\/ijms18122709","article-title":"Heat Shock Proteins\nin Vascular Diabetic\nComplications: Review and Future Perspective","volume":"18","author":"Bellini","year":"2017","journal-title":"Int. J. Mol. Sci."},{"issue":"1","key":"2026080408074318500_cit25","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1007\/s12035-012-8307-4","article-title":"From Mitochondrial Dysfunction to\nAmyloid Beta Formation: Novel Insights into the Pathogenesis of Alzheimer\u2019s\nDisease","volume":"46","author":"Leuner","year":"2012","journal-title":"Mol. Neurobiol."},{"issue":"5","key":"2026080408074318500_cit26","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1038\/s41583-020-0292-4","article-title":"Circuits and\nfunctions of the lateral habenula in health\nand in disease","volume":"21","author":"Hu","year":"2020","journal-title":"Nat. Rev. Neurosci."},{"key":"2026080408074318500_cit27","doi-asserted-by":"publisher","DOI":"10.3389\/fnbeh.2013.00017","article-title":"Genetic dissection of medial habenula-interpeduncular\nnucleus pathway function in mice","volume":"7","author":"Kobayashi","year":"2013","journal-title":"Front. Behav.\nNeurosci."},{"issue":"19","key":"2026080408074318500_cit28","doi-asserted-by":"publisher","first-page":"R873","DOI":"10.1016\/j.cub.2016.08.051","article-title":"The habenula","volume":"26","author":"Namboodiri","year":"2016","journal-title":"Curr. Biol."},{"key":"2026080408074318500_cit29","doi-asserted-by":"publisher","first-page":"130","DOI":"10.1016\/j.semcdb.2017.08.019","article-title":"Information processing\nin the vertebrate habenula","volume":"78","author":"Fore","year":"2018","journal-title":"Semin. Cell Dev.\nBiol."},{"issue":"6","key":"2026080408074318500_cit30","doi-asserted-by":"publisher","first-page":"786","DOI":"10.1111\/jnc.15771","article-title":"Translational patterns\nof ionotropic glutamate and GABA receptors during brain development\nand behavioral stimuli revealed by polysome profiling","volume":"164","author":"Peng","year":"2023","journal-title":"J. Neurochem."},{"key":"2026080408074318500_cit31","doi-asserted-by":"publisher","DOI":"10.3389\/fchem.2022.1041961","article-title":"Overview of myelin,\nmajor myelin lipids, and myelin-associated\nproteins","volume":"10","author":"Kister","year":"2023","journal-title":"Front. Chem."},{"issue":"18","key":"2026080408074318500_cit32","doi-asserted-by":"publisher","first-page":"6391","DOI":"10.1523\/JNEUROSCI.6221-11.2012","article-title":"Genomic\nAnalysis of Reactive Astrogliosis","volume":"32","author":"Zamanian","year":"2012","journal-title":"J. Neurosci."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c01074","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/30\/22017\/66238525\/acs.analchem.6c01074.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/30\/22017\/66238525\/acs.analchem.6c01074.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T12:23:10Z","timestamp":1785846190000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/30\/22017\/5231298\/Formic-Acid-Enhanced-Single-Pot-Sample-Preparation"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,23]]},"references-count":32,"journal-issue":{"issue":"30","published-online":{"date-parts":[[2026,7,23]]},"published-print":{"date-parts":[[2026,8,4]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c01074","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,23]]}}}