{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T14:58:32Z","timestamp":1791385112607,"version":"4.3.3"},"reference-count":30,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01GM127267"],"award-info":[{"award-number":["R01GM127267"]}],"id":[{"id":"10.13039\/100000057","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>Hydroxyl radical protein footprinting (HRPF) is a method for protein higher-order structure analysis that oxidizes solvent-accessible side chains of amino acids. One major limitation of HRPF is that results represent an ensemble average of all conformers in solution, preventing resolution of conformationally heterogeneous systems. Here, we established an inline hydrophobic interaction chromatography (HIC)-HRPF workflow that enables conformer-specific labeling while correcting for gradient-dependent differences in radical scavenging. Significant radical scavenging disparity exists between ammonium sulfate and phosphate buffers, and we compensated for this with 7 mM HEPES supplementation, restoring effective radical dose parity across the gradient. Using apo and holomyoglobin as a benchmark, we achieved baseline chromatographic separation of conformers. Multi-dose HRPF revealed conformer-specific oxidation patterns: apomyoglobin displayed enhanced labeling in peptide 81\u201397, specifically residue H83, consistent with destabilization of the F-helix and opening of the heme pocket, while holomyoglobin exhibited increased oxidation in peptide 120\u2013154 corresponding to M132, E137, and L138, suggesting hinge-like rearrangements in the H-helix upon heme incorporation. These results validate inline HIC-HRPF as a robust, conformer-specific probe of higher-order structure and highlight the necessity of scavenging parity across the gradient for reliable structural interpretation. The method establishes a framework for extending HRPF to dynamic conformationally heterogeneous systems resolvable by HIC.<\/jats:p>","DOI":"10.1021\/jasms.6c00291","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T09:01:28Z","timestamp":1789117288000},"page":"2542-2551","source":"Crossref","is-referenced-by-count":0,"title":["Inline HIC-HRPF Enables Conformer-Specific Structural\nAnalysis of Apo and Holomyoglobin"],"prefix":"10.1021","volume":"37","author":[{"given":"Godson I.","family":"Orachor","sequence":"first","affiliation":[{"name":"University of Mississippi , , , ,","place":["University, Mississippi, United States, 38677"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ajay","family":"Sharma","sequence":"additional","affiliation":[{"name":"University of Mississippi , , , ,","place":["University, Mississippi, United States, 38677"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9165-3987","authenticated-orcid":true,"given":"Sandeep K.","family":"Misra","sequence":"additional","affiliation":[{"name":"University of Mississippi , , , ,","place":["University, Mississippi, United States, 38677"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0115-0276","authenticated-orcid":true,"given":"Joshua S.","family":"Sharp","sequence":"additional","affiliation":[{"name":"University of Mississippi , , , ,","place":["University, Mississippi, United States, 38677"]},{"name":"University of Mississippi , , , ,","place":["University, Mississippi, United States, 38677"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"issue":"8","key":"2026100710064166800_cit1","doi-asserted-by":"publisher","first-page":"7532","DOI":"10.1021\/acs.chemrev.1c00432","article-title":"Hydroxyl Radical Protein Footprinting:\nA Mass Spectrometry-Based Structural Method for Studying the Higher\nOrder Structure of Proteins","volume":"122","author":"McKenzie-Coe","year":"2022","journal-title":"Chem. Rev"},{"issue":"1","key":"2026100710064166800_cit2","doi-asserted-by":"publisher","first-page":"311","DOI":"10.1021\/ac102426d","article-title":"Fast Photochemical\nOxidation of Proteins for Comparing Structures of Protein\u2013Ligand\nComplexes: The Calmodulin\u2013Peptide Model System","volume":"83","author":"Zhang","year":"2011","journal-title":"Anal. Chem"},{"issue":"1","key":"2026100710064166800_cit3","doi-asserted-by":"publisher","first-page":"4552","DOI":"10.1038\/s41598-017-04689-3","article-title":"Quantitative Protein\nTopography Measurements by High Resolution Hydroxyl Radical Protein\nFootprinting Enable Accurate Molecular Model Selection","volume":"7","author":"Xie","year":"2017","journal-title":"Sci. Rep"},{"issue":"1","key":"2026100710064166800_cit4","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1016\/j.bpj.2014.11.013","article-title":"Quantitative mapping\nof protein structure by hydroxyl radical footprinting-mediated structural\nmass spectrometry: A protection factor analysis","volume":"108","author":"Huang","year":"2015","journal-title":"Biophys. J"},{"issue":"1\u20133","key":"2026100710064166800_cit5","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.ijms.2006.08.018","article-title":"Laser flash photochemical oxidation\nto locate heme binding and conformational\nchanges in myoglobin","volume":"259","author":"Hambly","year":"2007","journal-title":"Int. J. Mass Spectrom"},{"issue":"3","key":"2026100710064166800_cit6","doi-asserted-by":"publisher","first-page":"614","DOI":"10.1006\/bbrc.2001.5628","article-title":"Unfolding\nof apomyoglobin examined by synchrotron footprinting","volume":"287","author":"Chance","year":"2001","journal-title":"Biochem. Biophys. Res. Commun"},{"issue":"7","key":"2026100710064166800_cit7","doi-asserted-by":"publisher","first-page":"3510","DOI":"10.1021\/acs.analchem.0c04872","article-title":"Inline\nLiquid Chromatography-Fast\nPhotochemical Oxidation of Proteins for Targeted Structural Analysis\nof Conformationally Heterogeneous Mixtures","volume":"93","author":"Tadi","year":"2021","journal-title":"Anal. Chem"},{"key":"2026100710064166800_cit8","doi-asserted-by":"publisher","DOI":"10.3791\/61861","article-title":"Laser-free\nHydroxyl\nRadical Protein Footprinting to Perform Higher Order Structural Analysis\nof Proteins","author":"Weinberger","year":"2021","journal-title":"J. Vis. Exp"},{"issue":"3","key":"2026100710064166800_cit9","doi-asserted-by":"publisher","first-page":"476","DOI":"10.1021\/jasms.3c00368","article-title":"Evaluating\nMass Spectrometry-Based\nHydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation\nSystem against a Synchrotron X-ray Beamline","volume":"35","author":"Jain","year":"2024","journal-title":"J. Am. Soc. Mass Spectrom"},{"issue":"51","key":"2026100710064166800_cit10","doi-asserted-by":"publisher","first-page":"18017","DOI":"10.1021\/acs.analchem.2c04365","article-title":"Structural Analysis\nof Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using\nFlash Oxidation Coupled In-Line with Ion Exchange Chromatography","volume":"94","author":"Cheng","year":"2022","journal-title":"Anal. Chem"},{"key":"2026100710064166800_cit11","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/j.jpba.2015.02.037","article-title":"Ion-exchange\nchromatography for the characterization of biopharmaceuticals","volume":"113","author":"Fekete","year":"2015","journal-title":"J. Pharm. Biomed. Anal"},{"key":"2026100710064166800_cit12","doi-asserted-by":"publisher","first-page":"130","DOI":"10.1016\/j.jchromb.2017.02.017","article-title":"Charge variants characterization\nof a monoclonal antibody by ion exchange chromatography coupled on-line\nto native mass spectrometry: Case study after a long-term storage\nat +5 \u00b0C","volume":"1048","author":"Leblanc","year":"2017","journal-title":"J. Chromatogr. B"},{"key":"2026100710064166800_cit13","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1016\/S0076-6879(09)63025-1","article-title":"Theory\nand use of hydrophobic interaction chromatography in protein purification\napplications","volume":"463","author":"McCue","year":"2009","journal-title":"Methods Enzymol"},{"issue":"21","key":"2026100710064166800_cit14","doi-asserted-by":"publisher","first-page":"12625","DOI":"10.1021\/acs.analchem.8b02787","article-title":"Real Time\nNormalization of Fast Photochemical Oxidation of Proteins Experiments\nby Inline Adenine Radical Dosimetry","volume":"90","author":"Sharp","year":"2018","journal-title":"Anal. Chem"},{"issue":"4","key":"2026100710064166800_cit15","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1006\/jmbi.1996.0596","article-title":"Is apomyoglobin\na molten globule? Structural characterization\nby NMR","volume":"263","author":"Eliezer","year":"1996","journal-title":"J. Mol. Biol"},{"issue":"6","key":"2026100710064166800_cit16","doi-asserted-by":"publisher","first-page":"1572","DOI":"10.1110\/ps.04635304","article-title":"Modulation of the structural\nintegrity\nof helix F in apomyoglobin by single amino acid replacements","volume":"13","author":"Picotti","year":"2004","journal-title":"Protein Sci"},{"issue":"1","key":"2026100710064166800_cit17","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1016\/0022-2836(86)90470-5","article-title":"X-ray structure\nand refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 \u00c5 resolution","volume":"192","author":"Kuriyan","year":"1986","journal-title":"J. Mol. Biol"},{"issue":"11","key":"2026100710064166800_cit18","doi-asserted-by":"publisher","first-page":"989","DOI":"10.1016\/j.tibs.2022.05.008","article-title":"Hydroxyl radical protein footprinting\nfor analysis\nof higher order structure","volume":"47","author":"Johnson","year":"2022","journal-title":"Trends Biochem. Sci"},{"issue":"5","key":"2026100710064166800_cit19","doi-asserted-by":"publisher","first-page":"1155","DOI":"10.1021\/jasms.0c00457","article-title":"Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in\nFast Photochemical Oxidation of Proteins Experiments","volume":"32","author":"Cheng","year":"2021","journal-title":"J. Am. Soc. Mass Spectrom"},{"issue":"16","key":"2026100710064166800_cit20","doi-asserted-by":"publisher","first-page":"6563","DOI":"10.1021\/ac901054w","article-title":"Fast photochemical\noxidation of protein footprints faster than protein unfolding","volume":"81","author":"Gau","year":"2009","journal-title":"Anal. Chem"},{"issue":"2","key":"2026100710064166800_cit21","doi-asserted-by":"publisher","first-page":"513","DOI":"10.1063\/1.555805","article-title":"Critical Review\nof rate constants for reactions of hydrated electrons, hydrogen atoms\nand hydroxyl radicals (\u00b7OH\/\u00b7O\u2013 in Aqueous Solution","volume":"17","author":"Buxton","year":"1988","journal-title":"J. Phys. Chem. Ref. Data"},{"key":"2026100710064166800_cit22","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1016\/0734-9750(89)90901-4","article-title":"Hydrophobic interaction\nchromatography","volume":"7","author":"Roettger","year":"1989","journal-title":"Biotechnol. Adv"},{"issue":"1","key":"2026100710064166800_cit23","doi-asserted-by":"publisher","first-page":"452","DOI":"10.1038\/s42003-022-03411-y","article-title":"Validated determination\nof NRG1 Ig-like domain structure by mass\nspectrometry coupled with computational modeling","volume":"5","author":"Khaje","year":"2022","journal-title":"Commun. Biol."},{"issue":"8","key":"2026100710064166800_cit24","doi-asserted-by":"publisher","first-page":"6045","DOI":"10.1021\/acs.analchem.5c06463","article-title":"Multidose Inline\nSize-Exclusion Chromatography\u2013Flash Oxidation for Structural\nAnalysis of Dynamic Protein\u2013Ligand Interactions of Antithrombin\nIII and Unfractionated Heparin","volume":"98","author":"Sharma","year":"2026","journal-title":"Anal. Chem"},{"issue":"16","key":"2026100710064166800_cit25","doi-asserted-by":"publisher","first-page":"6376","DOI":"10.1021\/ac070724u","article-title":"Effects of\nProtein Concentration\non the Extent of \u03b3-Ray-Mediated Oxidative Labeling Studied by\nElectrospray Mass Spectrometry","volume":"79","author":"Tong","year":"2007","journal-title":"Anal. Chem"},{"issue":"3","key":"2026100710064166800_cit26","doi-asserted-by":"publisher","first-page":"1121","DOI":"10.1021\/acs.analchem.3c04011","article-title":"Selectivity\nand Resolving Power of Hydrophobic Interaction Chromatography Targeting\nthe Separation of Monoclonal Antibody Variants","volume":"96","author":"Ewonde","year":"2024","journal-title":"Anal. Chem"},{"issue":"1","key":"2026100710064166800_cit27","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/s0021-9673(02)01077-4","article-title":"Hydrophobic\nInteraction Chromatography of Proteins. I. Comparison of Selectivity","volume":"972","author":"Machold","year":"2002","journal-title":"J. Chromatogr. A"},{"key":"2026100710064166800_cit28","doi-asserted-by":"publisher","first-page":"100020","DOI":"10.1016\/j.btecx.2020.100020","article-title":"Hydrophobic\nInteraction Chromatography as Polishing Step Enables Obtaining Ultra-Pure\nRecombinant Antibodies","volume":"324S","author":"Pereira Bresolin","year":"2020","journal-title":"J. Biotechnol"},{"key":"2026100710064166800_cit29","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/j.jchromb.2016.06.049","article-title":"Analysis of\nantibody-drug conjugates\nby comprehensive on-line two-dimensional hydrophobic interaction chromatography\nx reversed phase liquid chromatography hyphenated to high resolution\nmass spectrometry. II. Identification of sub-units for the characterization\nof even and odd load drug species","volume":"1032","author":"Sarrut","year":"2016","journal-title":"J. Chromatogr\nB Analyt Technol. Biomed Life Sci"},{"key":"2026100710064166800_cit30","doi-asserted-by":"publisher","first-page":"113703","DOI":"10.1016\/j.jpba.2020.113703","article-title":"A Hydrophobic Interaction Chromatography\nMethod Suitable for Quantitating\nIndividual Monoclonal Antibodies Contained in Co-Formulated Drug Products","volume":"193","author":"Luo","year":"2021","journal-title":"J. Pharm. Biomed Anal."}],"container-title":["Journal of the American Society for Mass Spectrometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jasms.6c00291","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/10\/2542\/68480228\/jasms.6c00291.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/10\/2542\/68480228\/jasms.6c00291.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T14:26:21Z","timestamp":1791383181000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jamsef\/article\/37\/10\/2542\/5428165\/Inline-HIC-HRPF-Enables-Conformer-Specific"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,11]]},"references-count":30,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,11]]},"published-print":{"date-parts":[[2026,10,7]]}},"URL":"https:\/\/doi.org\/10.1021\/jasms.6c00291","relation":{},"ISSN":["1044-0305","1879-1123"],"issn-type":[{"value":"1044-0305","type":"print"},{"value":"1879-1123","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,11]]}}}