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Ion Mobility Spectrometry Coupled to Mass Spectrometry for Glycoanalysis: Ion-Neutral Collision Cross Section Calibration and Application to Glycopeptides

Abby S Gelb, University of Nebraska - Lincoln


Glycoconjugates are central to various cellular processes, which when perturbed, may lead to dysfunction and disease. Specifically, protein glycosylation plays several key roles including cell-cell signaling, intermolecular recognition, protein folding, stability, and solubility. Protein glycosylation is non-template driven, and altered glycosylation may be influenced by the availability of nucleotide sugar donors and the regulation of various glycosidases and glycosyltransferases. Therefore, investigation of protein glycosylation is of high importance. Ion mobility spectrometry (IMS) is a gas-phase separation technique that allows for rapid separation (millisecond time scale) coupled to mass spectrometry (MS). IMS analysis provides arrival time distributions (ATDs) and subsequent collision cross section (CCS) measurements, which become analytically useful when comparing data to other laboratories. IMSMS has shown significant potential in the field of glycoanalysis. Specifically, traveling wave ion mobility spectrometry (TWIMS), commercialized in the Waters Synapt HDMS instrument, allows for traditional IMSMS analysis in addition to multiple dissociation techniques for a more enriched analysis. However, TWIMS CCS measurements are more complicated to calculate as the electric fields (EFs) are more complex than traditional IMS instrumentation, resulting in the need for calibration. This dissertation describes the further development of TWIMS calibration and its implications in measuring CCSs of glycopeptides, as well as first insights into the unknown glycosylation of a prostate cancer disease-related glycoprotein, human Hyaluronidase 1 (Hyal1). Chapter 1 provides an introduction into the complexity of protein glycosylation, as well as an overview of IMS techniques and their applications in glycoanalysis. Chapter 2 describes new insights into TWIMS CCS calibration as they relate to carbohydrates and peptides. Chapter 3 is an update on TWIMS CCS calibration to investigate a larger number of biomolecule classes. Chapter 4 provides CCS measurements of N-linked glycopeptides, where charge plays a key role in the overall structure. Chapter 5 completes the first step towards glycoproteomic profiling of Hyal1, where glycosylation is relatively unknown. As a whole, protein glycosylation is investigated using several techniques coupled to MS, resulting in the maturation of TWIMS CCS calibration and glycoproteomics analyses.^

Subject Area

Analytical chemistry

Recommended Citation

Gelb, Abby S, "Ion Mobility Spectrometry Coupled to Mass Spectrometry for Glycoanalysis: Ion-Neutral Collision Cross Section Calibration and Application to Glycopeptides" (2018). ETD collection for University of Nebraska - Lincoln. AAI10793239.