Glycoproteins are implicated in many diseases including HIV/AIDS or cancer. Investigating how the chemical and conformational heterogeneities of the carbohydrate moieties modulate the three-dimensional structure in a glycoprotein is crucial for designing potential antibody-based vaccines. However, structural analysis of glycoproteins by conventional biophysics methods such as NMR spectroscopy and crystallography is very challenging.
We developed a tandem-trapped ion mobility spectrometer – mass spectrometer (tTIMS/MS), consisting of two TIMS analyzers embedded in a ESI-QqTOF mass spectrometer. tTIMS/MS couples the ability of TIMS to retain native-like structures at high resolving powers with the ability to mobility-select and to energetically activate ions prior to conducting ion mobility measurement in the second TIMS analyzer. Additionally, ions with specific m/z range can be isolated in the quadrupole and activated in the collision cell, allowing MS³ experiments of mobility- and m/z-selected ions.
We employ tTIMS/MS to investigate avidin, a glycoprotein which exists as homotetramers with 128 amino acids in each subunit and an N-glycan attached to Asn17. Mobility selection and collisional activation experiments at low voltages show that the native avidin tetramers consist of unresolved, structurally distinct, non-interconverting species, with solvent adducts buried within binding interfaces/pockets. CID of avidin tetramers at high voltages in tTIMS generates compact monomers, dimers, and trimers, revealing the subunit topology. We identify various glycoforms and glycoforms combinations in avidin monomers and oligomers. Finally, we perform top-down sequencing of mobility-selected tetrameric avidin by CID in tTIMS. Distinct avidin monomers generated by CID are then isolated in the quadrupole and exposed to a second ion activation in the collision cell. The obtained mass spectrum shows fragments of avidin base and the attached N-glycans. Our results demonstrate the ability of tTIMS/MS to study the primary- and higher order structures of glycoprotein complexes. Further advances in glycoprotein structural studies employing a newly-constructed orthogonal configuration of tTIMS/MS is currently underway.

Mark Ridgeway

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