Differential Melting Voltage by Tandem-Trapped Ion Mobility Spectrometry: Glycan Branching Influences Glycoprotein Stability

Glycoproteins encompass ~50-70% of all human proteins, and as such, are involved in many aspects of cellular processes. It is crucial to study how distinct glycan molecules modulate the biophysical properties and the function of the protein. However, investigating the full set of protein glycoforms remains challenging due to the difficulty in separating distinct protein glycoforms. In this study, we developed a method based on tandem-trapped ion mobility/tandem-mass spectrometry to characterize the relative stability of distinct protein glycoforms, termed Differential Melting Voltage. We applied this method to characterize the full set of Ribonuclease B glycoforms, and obtained several key insights: (1) there is a non-linear relationship between glycoform stability and both the mass of the attached glycans and the overall size of the protein, and (2) the branching pattern of the attached glycan possibly plays a role in modulating protein stability. These results highlight the potential of the Differential Melting Voltage approach for resolving subtle structural and stability differences among closely related protein glycoforms.