Protein sialylation has been linked to health and many diseases and is widely involved in pharmaceuticals in an isomer- or terminal structure-sensitive manner. Sialylation is often present in a large portion (~80%) of glycoproteins which represents a unique terminal glycosylation form with negatively charged, acidic residues and often with five or more hydroxyl groups and one carboxyl group in the terminal glycans. However, to investigate the effect of sialylation on glycoprotein structures, traditional methods either require tedious glycan release-chemical derivatization processes and concomitant large sample loss or need to denature the target protein before sialylation information can be obtained. These procedures often lead to the loss of information about structural role of sialylation in regulating protein folding/unfolding and their topologies. Here, we modify a native ion mobility-mass spectrometric approach to elucidate the effects of two kinds of terminal sialylation on two important carrier glycoproteins, fetuin and transferrin. In our approach,“zero charge selection” allows all forms of sialylated glycoprotein ions produced by nano-electrospray ionization to be simultaneously activated in a charge state-independent regime, where dispersive energy is sequentially enforced to allow simultaneous stepwise unfolding and sialic acid release. Driftscope traces depict the overall all ion unfolding/fragmentation (AIU/AIF) spectrum. CIUSuite is utilized to visualize and quantitatively characterize the stepwise unfolding pathway whereby ions with all charge states are included. UniDec is employed to track and visualize the gradual sialylation release process which involves all ion deconvolution.

Ion mobility-mass spectrometry reveals the effect of sialylation on glycoprotein structures

2019 Fall National Meeting

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