“Mass Spectrometry, Ion Mobility, and Ion Chemistry: Tools for Characterizing the Structures of Proteins in Solution and in the Gas Phase”
Professor Matt Bush, University of Washington, Seattle, Washington
Native mass spectrometry is an emerging approach for characterizing the stoichiometry, assembly, and shapes of protein complexes in solution. Native mass spectrometry is especially useful for investigating proteins and protein complexes that are challenging to characterize using condensed-phase experiments, including those that are heterogeneous, have large mass, and are membrane bound. I will discuss how my lab uses mass spectrometry, ion mobility, and ion chemistry to probe the formation of gas-phase ions, the effects of charge on the structures of biological molecules in the absence of solvent, and the structures and dynamics of proteins and protein complexes in solution.
“Application and Development of a Hydrophobic Tagging Strategy to Quantify Ovarian Cancer Modulation of Secreted N-linked Glycans”
Elizabeth Hecht, Laboratory of Professor David Muddiman, N.C. State University, Department of Chemistry
Derivatization strategies for mass spectrometry (MS) can accommodate detection of low-abundant species and confer quantitative analysis. N-linked glycans, a co-translational protein modification, are a species of interest for cancer research, yet their hydrophilicity limits their electrospray ionization (ESI) efficiency. In 2011, our group developed the individuality normalization when labeling with isotopic glycan hydrazide tags (INLIGHT) strategy, which facilitated ESI-MS glycomics and for the first time allowed for highly sensitive, quantitative studies to be executed.
Glycan biomarkers have a particular interest for ovarian cancer (OVC) research. A plasma repository spanning the four stages of OVC was curated and the relative abundances of 63 glycans between matched cancer and control patients were calculated. These glycan cancer burden ratios were modeled using stepwise forward regression techniques against terms related to characteristics of the patient samples, cancer, and glycans. Contradicting recent literature reports, it was determined that the chemical class of glycans was not significant. The compositions of seven of N-glycans and four N-glycans were determined to be statistically significant after Bonferroni (p < 0.05) and false-discovery-rate (p ≤ 0.05) corrections, respectively. The abundances of all significant glycans showed a novel trend with OVC stage, sequentially reversing in magnitudes and abundances.
The power of MS studies increases with the vastly improved detection limits of mass analyzers and the number of biological samples. First, design of experiments was used to optimize the ESI Q ExactiveTM HF parameters for hydrophobic-tagged glycans, resulting in an average 2-fold increase in abundance, detailing new parameter interactions, and minimizing saccharide gas phase dissociation. Second, filter aided N-glycan separation (FANGS) was evolved to purify glycans from plasma glycoproteins and quantitatively compared to solid phase extraction. The abundances and identities of glycans between the two workflows were not statistically significantly different, thus a new purification method was developed for high-throughput glycomics.