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It requires high\u2010resolution separations of histone proteoforms before MS and tandem MS (MS\/MS) analysis. In this work, for the first time, we combined SDS\u2010PAGE\u2010based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI\u2010MS) with capillary zone electrophoresis (CZE)\u2010MS\/MS for high\u2010resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS\u2010PAGE gel and follow\u2010up cleanup as well as CZE\u2010MS\/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high\u2010resolution separation and characterization of histone proteoforms. SDS\u2010PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high\u2010resolution separations of SDS\u2010PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.<\/jats:p>","DOI":"10.1002\/pmic.202300650","type":"journal-article","created":{"date-parts":[[2024,7,17]],"date-time":"2024-07-17T12:59:45Z","timestamp":1721221185000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Combining SDS\u2010PAGE to capillary zone electrophoresis\u2010tandem mass spectrometry for high\u2010resolution top\u2010down proteomics analysis of intact histone proteoforms"],"prefix":"10.1002","volume":"24","author":[{"given":"Fei","family":"Fang","sequence":"first","affiliation":[{"name":"Department of Chemistry Michigan State University  East Lansing Michigan 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