Protein structure characterization facilities mechanistic understanding of protein function. However, analyzing heterogeneous conformations, proteoforms and protein–ligand interactions can be very challenging for traditional biophysics tools.<br/>We recently developed a tandem-trapped ion mobility spectrometer – mass spectrometer (tTIMS/MS) which consists of two TIMS analyzers embedded in an ESI-QqTOF mass spectrometer. tTIMS/MS is capable of retaining nativelike structures of proteins and protein complexes when “native” ESI conditions and “soft” instrumental parameters are employed. The interface region between the two TIMS analyzer allows mobility-selection and collisional-activation of protein conformations, enabling us to characterize their relative stabilities.<br/>Here, we discuss how tTIMS/MS can be used to characterize the influence of glycans on the stability and ligand-binding strength of proteins. To this end, we use ribonuclease (RNase) A, its glycosylated form RNase B and RNase-nucleotide complexes as model systems.<br/>First and foremost, we demonstrate how nativelike, heterogeneous structures of RNase A, RNase B, and RNase A-nucleotide complexes are preserved using tTIMS/MS. Then, the sets of methods that we established to characterize the structure, binding affinity and gas-phase stability of RNase A-nucleotide complexes are discussed comprehensively. At the end, the parallel experiments carried out for RNase B are compared with those for RNase A. The preliminary results indicate that glycosylation stabilizes the RNase against unfolding; stabilizes the RNase-nucleotide complexes from losing ligand.

Towards structure elucidation of proteins, glycoproteins and protein-ligand complexes using tandem trapped ion mobility spectrometry-mass spectrometry

ACS Spring Meeting 2021

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