{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T15:01:02Z","timestamp":1791385262435,"version":"4.3.3"},"reference-count":56,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["HL109810"],"award-info":[{"award-number":["HL109810"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["HL181422"],"award-info":[{"award-number":["HL181422"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000179","name":"Office of the Director","doi-asserted-by":"publisher","award":["S10 OD018475"],"award-info":[{"award-number":["S10 OD018475"]}],"id":[{"id":"10.13039\/100000179","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Top-down proteomics (TDP) enables comprehensive characterization of intact proteoforms, but the analysis of membrane proteoforms remains challenging because of their hydrophobicity and relatively low abundance. To address these challenges, we previously developed Azo, a photocleavable surfactant that enables efficient solubilization and top-down analysis of membrane proteins. Subsequently, we established an Azo-enabled TDP method permitting the comprehensive characterization of phospholamban (PLN), a transmembrane protein that plays crucial roles in calcium handling, directly from cardiac tissue. However, the original workflow required at least 10 mg of human cardiac tissue, making it unsuitable for biopsy-scale and other sample-limited studies. Herein, we developed a streamlined Azo-enabled one-pot extraction strategy for high-sensitivity top-down analysis of PLN that reduces the required tissue input to 1 mg while preserving analytical performance. Using online liquid chromatography\u2013tandem mass spectrometry, we achieved highly reproducible detection, quantification, and post-translational modification localization of endogenous PLN proteoforms from 1 mg of human cardiac tissue. Notably, the streamlined strategy reduced the tissue required for PLN protein extraction 10-fold while retaining the same capacity for comprehensive characterization in the one-pot extraction. Collectively, these results establish a practical strategy for TDP of PLN proteoforms from biopsy-scale and other sample-limited myocardial specimens.<\/jats:p>","DOI":"10.1021\/jasms.6c00309","type":"journal-article","created":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T15:02:08Z","timestamp":1789830128000},"page":"2273-2280","source":"Crossref","is-referenced-by-count":0,"title":["A Streamlined One-Pot\nExtraction Method Enables High-Sensitivity\nTop-Down Proteomic Characterization of Phospholamban from Biopsy-Scale\nCardiac Tissue"],"prefix":"10.1021","volume":"37","author":[{"given":"Isabella R.","family":"Clemmer","sequence":"first","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0870-0127","authenticated-orcid":true,"given":"Holden T.","family":"Rogers","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2852-125X","authenticated-orcid":true,"given":"Mallory C.","family":"Wilson","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhan","family":"Gao","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53705"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4488-5053","authenticated-orcid":true,"given":"Hsin-Ju","family":"Chan","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zachery R.","family":"Gregorich","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53705"]},{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farhan","family":"Raza","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53705"]},{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5211-6812","authenticated-orcid":true,"given":"Ying","family":"Ge","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]},{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53705"]},{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]},{"name":"University of Wisconsin-Madison , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,19]]},"reference":[{"issue":"6380","key":"2026100710073186900_cit1","doi-asserted-by":"publisher","first-page":"1106","DOI":"10.1126\/science.aat1884","article-title":"Proteoforms as the\nnext Proteomics Currency","volume":"359","author":"Smith","year":"2018","journal-title":"Science"},{"issue":"3","key":"2026100710073186900_cit2","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1038\/nmeth.2369","article-title":"Proteoform: A Single\nTerm Describing Protein Complexity","volume":"10","author":"Smith","year":"2013","journal-title":"Nat. Methods"},{"issue":"1","key":"2026100710073186900_cit3","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1146\/annurev-anchem-071015-041550","article-title":"Progress\nin Top-Down Proteomics and\nthe Analysis of Proteoforms","volume":"9","author":"Toby","year":"2016","journal-title":"Annu. Rev. Anal.\nChem. Palo Alto Calif"},{"key":"2026100710073186900_cit4","doi-asserted-by":"publisher","first-page":"102180","DOI":"10.1016\/j.cbpa.2022.102180","article-title":"Deciphering\nCombinatorial Post-Translational Modifications by Top-down Mass Spectrometry","volume":"70","author":"Brodbelt","year":"2022","journal-title":"Curr. Opin. Chem. Biol."},{"issue":"1","key":"2026100710073186900_cit5","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1002\/mas.21348","article-title":"Top-down Mass Spectrometry for the Analysis of Combinatorial\nPost-Translational Modifications","volume":"32","author":"Lanucara","year":"2013","journal-title":"Mass Spectrom.\nRev."},{"issue":"11","key":"2026100710073186900_cit6","doi-asserted-by":"publisher","first-page":"196A","DOI":"10.1021\/ac0415657","article-title":"Peer Reviewed:\nTop-Down Proteomics","volume":"76","author":"Kelleher","year":"2004","journal-title":"Anal. Chem."},{"issue":"1","key":"2026100710073186900_cit7","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1021\/acs.analchem.7b04747","article-title":"Top-Down Proteomics:\nReady for Prime Time?","volume":"90","author":"Chen","year":"2018","journal-title":"Anal. 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Elegans Nematodes","volume":"62","author":"Leipert","year":"2023","journal-title":"Angew. Chem., Int. Ed."},{"issue":"19","key":"2026100710073186900_cit37","doi-asserted-by":"publisher","first-page":"e2222081120","DOI":"10.1073\/pnas.2222081120","article-title":"High Sensitivity\nTop\u2013down Proteomics Captures Single Muscle Cell Heterogeneity\nin Large Proteoforms","volume":"120","author":"Melby","year":"2023","journal-title":"Proc. Natl. Acad. Sci.\nU. S. 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Sci."},{"issue":"3","key":"2026100710073186900_cit40","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1006\/jmcc.1998.0897","article-title":"Reduced Ca(2+)-Sensitivity of SERCA\n2a in Failing Human Myocardium Due to Reduced Serin-16 Phospholamban\nPhosphorylation","volume":"31","author":"Schwinger","year":"1999","journal-title":"J. Mol. Cell. Cardiol."},{"issue":"32","key":"2026100710073186900_cit41","doi-asserted-by":"publisher","first-page":"26596","DOI":"10.1074\/jbc.M112.382713","article-title":"Lethal, Hereditary\nMutants of Phospholamban Elude Phosphorylation by Protein Kinase A","volume":"287","author":"Ceholski","year":"2012","journal-title":"J. Biol. Chem."},{"issue":"9","key":"2026100710073186900_cit42","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0106746","article-title":"Comparison\nof the Structure and Function of Phospholamban\nand the Arginine-14 Deficient Mutant Associated with Dilated Cardiomyopathy","volume":"9","author":"Hughes","year":"2014","journal-title":"PLoS One"},{"issue":"647","key":"2026100710073186900_cit43","doi-asserted-by":"publisher","DOI":"10.1126\/scisignal.aaz1436","article-title":"14\u20133-3\nBinding Creates a Memory of Kinase Action by Stabilizing the Modified\nState of Phospholamban","volume":"13","author":"Menzel","year":"2020","journal-title":"Sci. Signal."},{"issue":"5","key":"2026100710073186900_cit44","doi-asserted-by":"publisher","first-page":"784","DOI":"10.1016\/j.hrthm.2010.11.035","article-title":"CAMKII REGULATION OF PHOSPHOLAMBAN\nAND SR Ca2+ LOAD","volume":"8","author":"Mattiazzi","year":"2011","journal-title":"Heart Rhythm Off. J. Heart\nRhythm Soc."},{"issue":"13","key":"2026100710073186900_cit45","doi-asserted-by":"publisher","first-page":"3265","DOI":"10.1021\/ja973478k","article-title":"Electron\nCapture Dissociation of\nMultiply Charged Protein Cations. A Nonergodic Process","volume":"120","author":"Zubarev","year":"1998","journal-title":"J. Am. Chem. Soc."},{"issue":"19","key":"2026100710073186900_cit46","doi-asserted-by":"publisher","first-page":"4250","DOI":"10.1021\/ac990684x","article-title":"Localization of Labile Posttranslational\nModifications by Electron Capture Dissociation: The Case of \u03b3-Carboxyglutamic\nAcid","volume":"71","author":"Kelleher","year":"1999","journal-title":"Anal. Chem."},{"issue":"9","key":"2026100710073186900_cit47","doi-asserted-by":"publisher","first-page":"1805","DOI":"10.1007\/s13361-017-1710-3","article-title":"The Impact\nof Phosphorylation on Electron Capture Dissociation of Proteins: A\nTop-down Perspective","volume":"28","author":"Chen","year":"2017","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"1","key":"2026100710073186900_cit48","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1021\/acs.analchem.7b04810","article-title":"The Role of Electron Transfer Dissociation in Modern\nProteomics","volume":"90","author":"Riley","year":"2018","journal-title":"Anal. Chem."},{"issue":"34","key":"2026100710073186900_cit49","doi-asserted-by":"publisher","first-page":"12646","DOI":"10.1021\/ja4029654","article-title":"Complete Protein\nCharacterization Using Top-Down Mass\nSpectrometry and Ultraviolet Photodissociation","volume":"135","author":"Shaw","year":"2013","journal-title":"J. Am. Chem. 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