Linkage between Quaternary Structure and Subunit Sequencing of Zebra Finch Hemoglobin via Hyphenation of Size Exclusion Chromatography with Native Top-Down Mass Spectrometry.

Hemoglobin is the protein responsible for oxygen transport in many vertebrates, and any structural alteration can lead to health issues. Although well characterized in humans, it is less known for birds. Studies have reported the identification of several α subunits along with the presence of endogenous cofactors in different avian erythrocytes. However, the native structure of avian hemoglobin remains elusive. Native top-down mass spectrometry (nTD-MS) offers powerful insights into biomolecular complexes, providing information on quaternary structure, subunit connectivity, stoichiometry, and subunit sequences. However, most nTD-MS approaches use direct infusion, which can limit the multiprotein complex population characterization. Here, we report on the development of a size-exclusion-chromatography (SEC) nTD-MS approach, including different activation methods to characterize the zebra finch hemoglobin structure. Three different tetramer populations were separated and characterized using optimized pMS2 (pseudo-MS2, complex-up) workflows to induce subunit and endogenous cofactor release previously reported by our group. The analytical strategy was improved with the addition of an extra level of characterization through the implementation of a pMS3 (pseudo-MS3, complex-down) step with controlled pressure, allowing almost complete sequence coverage of all the subunits (>94%), along with the identification of inositol pentaphosphate as a cofactor of the tetramer structure. Altogether, these results pinpoint the key role of SEC-nTD-MS workflows to enable a complete structural characterization of hemoglobin complexes, which could provide crucial information regarding oxygen affinity, bird environment adaptation, or phylogeny.