What does this research mean for the field?

An inline hydrophobic interaction chromatography (HIC)-HRPF workflow enables conformer-specific structural analysis of conformationally heterogeneous protein systems by correcting for gradient-dependent differences in radical scavenging. Novelty: ClaimNovelty.METHODOLOGICAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

What question did this study set out to answer?

To establish an inline hydrophobic interaction chromatography-hydroxyl radical protein footprinting workflow that enables conformer-specific structural labeling of conformationally heterogeneous protein mixtures.

September 14, 2026Journal of the American Society for Mass Spectrometry

Inline HIC-HRPF Enables Conformer-Specific Structural Analysis of Apo and Holomyoglobin

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Authors

GOGodson I. Orachor ASAjay Sharma SMSandeep K. Misra

Discussion

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Overview

Experimental study demonstrates conformer-specific structural labeling of apo- and holomyoglobin via inline chromatography, establishing a framework to resolve heterogeneous protein mixtures.

Key Points

To establish an inline hydrophobic interaction chromatography-hydroxyl radical protein footprinting workflow that enables conformer-specific structural labeling of conformationally heterogeneous protein mixtures.
Coupled inline hydrophobic interaction chromatography (HIC) with hydroxyl radical protein footprinting (HRPF) to separate and label apo- and holomyoglobin conformers.
Supplemented elution buffers with 7 mM HEPES to compensate for radical scavenging disparities between ammonium sulfate and phosphate gradients.
Achieved baseline chromatographic separation of apomyoglobin and holomyoglobin conformers while maintaining radical dose parity across the gradient.
Identified enhanced oxidation in apomyoglobin at peptide 81–97, specifically residue H83, reflecting destabilization of the F-helix and opening of the heme pocket.
Observed increased labeling in holomyoglobin across peptide 120–154 at residues M132, E137, and L138, indicating H-helix hinge rearrangements upon heme incorporation.

Cite This Study

Orachor et al. (2026) studied this question.

synapsesocial.com/papers/6aa7b2f70926e14a848b1867 https://doi.org/10.1021/jasms.6c00291

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September 14, 2026Journal of the American Society for Mass Spectrometry

Inline HIC-HRPF Enables Conformer-Specific Structural Analysis of Apo and Holomyoglobin

View Full Paper

Ask AI

Bookmark

Authors

GOGodson I. Orachor ASAjay Sharma SMSandeep K. Misra

Discussion

Member takes

Overview

Experimental study demonstrates conformer-specific structural labeling of apo- and holomyoglobin via inline chromatography, establishing a framework to resolve heterogeneous protein mixtures.

Key Points

To establish an inline hydrophobic interaction chromatography-hydroxyl radical protein footprinting workflow that enables conformer-specific structural labeling of conformationally heterogeneous protein mixtures.
Coupled inline hydrophobic interaction chromatography (HIC) with hydroxyl radical protein footprinting (HRPF) to separate and label apo- and holomyoglobin conformers.
Supplemented elution buffers with 7 mM HEPES to compensate for radical scavenging disparities between ammonium sulfate and phosphate gradients.
Achieved baseline chromatographic separation of apomyoglobin and holomyoglobin conformers while maintaining radical dose parity across the gradient.
Identified enhanced oxidation in apomyoglobin at peptide 81–97, specifically residue H83, reflecting destabilization of the F-helix and opening of the heme pocket.
Observed increased labeling in holomyoglobin across peptide 120–154 at residues M132, E137, and L138, indicating H-helix hinge rearrangements upon heme incorporation.

Cite This Study

Orachor et al. (2026) studied this question.

synapsesocial.com/papers/6aa7b2f70926e14a848b1867 https://doi.org/10.1021/jasms.6c00291

View Full Paper

Ask AI

Bookmark