Session: Parallel session 9 - Top-down and Structural analysis
Development of innovative analytical strategies based on Top-Down Mass Spectrometry (TD-MS) for characterizing bird hemoglobins and it active form
Hemoglobin is the protein responsible for the blood oxygen transport in many vertebrates. Any abnormalities in hemoglobin structure, such as glycation, can lead to serious health outcomes. Conversely, blood glucose and blood hemoglobin concentrations have been shown to negatively correlate, giving rise to the bird paradox: “high glucose-low glycation-long lifespan”. Therefore, cutting-edge analytical strategies are needed to pinpoint the structural determinants responsible for glycation resistance in bird hemoglobin.
Top-down mass spectrometry (TD-MS) entails the fragmentation of intact biomolecules to cross-correlate the intact mass with the modifications of the aminoacids for an accurate proteoform description. This strategy can be taken one step further in combination with native MS (nMS) connecting structural information about the tertiary and quaternary structure of proteins with their sequence, bridging the gap between proteomics and structural biology. In this context, we combined size exclusion chromatography (SEC) with TD-MS using various fragmentation methods in native conditions to develop an innovative complex-down strategy to unveil for the first time the structure of Zebra Finch hemoglobin.
SEC separation put in evidence the co-existence of three tetrameric structures within the Zebra Finch hemoglobin sample. The subunit release of the complex was performed by increasing the voltage in the source of the mass spectrometer, pinpointing that structural differences between the three tetramers stemmed from the substitution of the aA to the aD globin unit. Further isolation and fragmentation of the subunits with HCD, ETD, and UVPD led to a complete sequencing providing more than 90% of sequence coverage.
This study highlights the perfect suitability of complex-down workflows to characterize different biologically-relevant systems, allowing to unravel structural insights that can be crucial for a better understanding of the structure-function relationship.
Top-down mass spectrometry (TD-MS) entails the fragmentation of intact biomolecules to cross-correlate the intact mass with the modifications of the aminoacids for an accurate proteoform description. This strategy can be taken one step further in combination with native MS (nMS) connecting structural information about the tertiary and quaternary structure of proteins with their sequence, bridging the gap between proteomics and structural biology. In this context, we combined size exclusion chromatography (SEC) with TD-MS using various fragmentation methods in native conditions to develop an innovative complex-down strategy to unveil for the first time the structure of Zebra Finch hemoglobin.
SEC separation put in evidence the co-existence of three tetrameric structures within the Zebra Finch hemoglobin sample. The subunit release of the complex was performed by increasing the voltage in the source of the mass spectrometer, pinpointing that structural differences between the three tetramers stemmed from the substitution of the aA to the aD globin unit. Further isolation and fragmentation of the subunits with HCD, ETD, and UVPD led to a complete sequencing providing more than 90% of sequence coverage.
This study highlights the perfect suitability of complex-down workflows to characterize different biologically-relevant systems, allowing to unravel structural insights that can be crucial for a better understanding of the structure-function relationship.