{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T12:18:08Z","timestamp":1780575488739,"version":"3.54.1"},"reference-count":47,"publisher":"Wiley","issue":"24","license":[{"start":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T00:00:00Z","timestamp":1762732800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35GM147397"],"award-info":[{"award-number":["R35GM147397"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteomics"],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:label\/>\n                    <jats:p>Proteoforms, the endogenous forms of proteins that take into account all sources of variation (genetic as well as co\u2010 and post\u2010translational), may be more relevant for understanding complex biological mechanisms, including disease phenotypes, than generically defined \u201cprotein families\u201d. Mass spectrometry\u2010based top\u2010down proteomics often utilizes electrophoretic techniques that rely on sodium dodecyl sulfate (SDS) to fractionate proteoforms prior to liquid chromatography\u2013tandem mass spectrometry (LC\u2010MS\/MS) analysis. Methanol\u2010chloroform\u2010water (MCW) precipitation is commonly utilized for SDS removal due to its efficacy and low cost, yet it may lead to poor recovery of smaller proteoforms. As a technical contribution to top\u2010down proteomics, four commercial SDS clean\u2010up alternatives were benchmarked against MCW. Results indicate that MCW yields fewer proteoform identifications, particularly among small and acidic proteoforms. The analysis of post\u2010translational modifications identified using the different clean\u2010up methods indicates increased prevalence of methylation modifications post\u2013MCW clean\u2010up. Among the commercial kits, DetergentOUT and HiPPR achieved SDS removal comparable to MCW but at a higher cost. For studies sensitive to loss of low molecular weight or acidic proteoforms, these kits may offer an advantage. Alternatively, at a lower cost, Minute SDS provides sufficient SDS removal and broader proteome coverage.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Summary<\/jats:title>\n                    <jats:p>This study aims to support the growing top\u2010down proteomics (TDP) community by addressing a practical challenge in sample preparation: efficient SDS removal following molecular weight\u2010based fractionation. As TDP gains traction for its ability to directly characterize proteoforms and their post\u2010translational modifications (PTMs), accessible and reliable workflows become increasingly important. Detergent removal is a key step, as SDS, though essential for protein solubilization and separation, interferes with mass spectrometry. Traditional methods like methanol\u2010chloroform\u2010water (MCW) precipitation can be labor\u2010intensive and may lead to loss of small proteoforms. In this work, we compare four commercially available SDS clean\u2010up kits to MCW based on SDS removal efficiency, proteoform recovery, PTM retention, and ease of use. Our findings provide practical guidance regarding each method's trade\u2010offs, aiming to inform more streamlined, reproducible TDP workflows. By benchmarking these kits, we contribute to ongoing efforts to democratize TDP and enhance its utility in biological and clinical research.<\/jats:p>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>The golden standard for SDS removal is methanol\u2010chloroform\u2010water (MCW) precipitation, which is labor\u2010intensive and, as demonstrated by both earlier reports and the present work, may lead to loss of small proteoforms.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>This study benchmarks four commercially available SDS clean\u2010up kits against MCW, considering SDS removal efficiency, proteoform recovery, retention of post\u2010translationally modified proteoforms, and ease of use.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Resin\u2010based clean\u2010up methods, DetergentOUT and HiPPR, were highly effective in SDS removal, and comparable to MCW clean\u2010up.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Minute SDS and SurfactAway kits, on the other hand, allowed the identification of a slightly higher number of proteoforms.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/pmic.70067","type":"journal-article","created":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T08:04:22Z","timestamp":1762761862000},"page":"30-41","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Comparative Analysis of Proteoform Clean\u2010Up Methods for In\u2010Depth Top\u2010Down Proteomics"],"prefix":"10.1002","volume":"25","author":[{"given":"Alyssa A.","family":"Williams","sequence":"first","affiliation":[{"name":"School of Biological Sciences University of Oklahoma  Norman Oklahoma USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kendalyn N.","family":"Firenza","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of Oklahoma  Norman Oklahoma USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy K.","family":"Carfagno","sequence":"additional","affiliation":[{"name":"School of Biological Sciences University of Oklahoma  Norman Oklahoma USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jake T.","family":"Kline","sequence":"additional","affiliation":[{"name":"School of Biological Sciences University of Oklahoma  Norman Oklahoma USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6334-1046","authenticated-orcid":false,"given":"Luca","family":"Fornelli","sequence":"additional","affiliation":[{"name":"School of Biological Sciences University of Oklahoma  Norman Oklahoma USA"},{"name":"Department of Chemistry and Biochemistry University of Oklahoma  Norman Oklahoma 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