. Protein glycosylation is a prevalent, chemically complex, and biologically diverse post-translational modification (PTM) involved in a wide array of cellular functions. Glycosylation plays essential roles in regulation of cellular proliferation and metabolic processes, and changes in glycosylation are universal features of malignant transformation and tumor progression. Metastasis, or the spread of cancer to non-adjacent parts of the body, is a particularly insidious characteristic of many aggressive cancers. Evidence suggests that specific changes in glycosylation may reflect fitness of tumor progression and metastatic potential, making global characterization of glycosylation crucial to understanding the molecular basis of cancer aggression/metastasis. Despite the critical importance of glycosylation in cancer research, current technology for characterizing this PTM is underdeveloped. Mass spectrometry (MS) is the gold standard for analysis of PTMs, but the chemical complexity of glycosylation has significantly slowed progress of MS technology relative to other modifications. This proposal introduces activated-ion electron transfer dissociation (AI-ETD) as a new tandem MS approach for comprehensive characterization of intact glycopeptides. The combination of infrared photo-activation and electron-driven radical fragmentation in AI-ETD generates peptide sequence and glycan composition information in a single tandem MS event. This eliminates multiple tandem MS scans per precursor that are necessary in current approaches, improving sensitivity and effectively doubling throughput. In this application, AI-ETD will be implemented on the newest generation of Orbitrap MS systems (Fusion Lumos) to capitalize on its robust data- acquisition platform, and high-throughput AI-ETD methods for glycoproteomic experiments will be developed. These methods will be utilized in glycoproteomic characterization of three isogenic human cancer cell lines that represent non-, intermediate-, and highly-metastatic forms of melanoma. By enabling characterization of hundreds of glycosites and thousands of glycans, this work will be the most comprehensive glycoproteomic comparison of cancer cell lines yet, allowing investigation of glycosylation signatures of cancer aggression with unprecedented breadth and depth across the glycoproteome. This data set will advance the understanding of cancer metastasis at a molecular level and reveal new insights into the role of glycosylation in cancer. Upon completion of this project, I will seek postdoctoral training in a preeminent cancer glycobiology laboratory, complementing the technology development focus of my graduate research. The combination of expertise in MS instrumentation development and the glycoscience of cancer will uniquely equip me for a prolific career as an independent scientist at the interface of biology and technology and the forefront of cancer research.

Public Health Relevance

. Altered protein glycosylation is a universal feature of cancer cells; as such, understanding the identities and molecular functions of glycosylation modifications is critical to advancing our knowledge of the molecular biology of cancer. Unfortunately, the chemical complexity and biological diversity of glycosylation makes it an especially challenging post-translational modification to characterize. This proposal centers on the development of new mass spectrometry instrumentation to significantly improve the analysis of protein glycosylation, with an ultimate goal of elucidating glycosylation signatures and biomarkers specific to cancer aggression and metastatic potential.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Project #
1F99CA212454-01
Application #
9229818
Study Section
Special Emphasis Panel (ZCA1-RTRB-R (A1))
Program Officer
Mcguirl, Michele
Project Start
2016-09-19
Project End
2018-08-31
Budget Start
2016-09-19
Budget End
2017-08-31
Support Year
1
Fiscal Year
2016
Total Cost
$33,141
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Riley, Nicholas M; Westphall, Michael S; Hebert, Alexander S et al. (2017) Implementation of Activated Ion Electron Transfer Dissociation on a Quadrupole-Orbitrap-Linear Ion Trap Hybrid Mass Spectrometer. Anal Chem 89:6358-6366
Riley, Nicholas M; Westphall, Michael S; Coon, Joshua J (2017) Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation. J Proteome Res 16:2653-2659
Rush, Matthew J P; Riley, Nicholas M; Westphall, Michael S et al. (2017) Sulfur Pentafluoride is a Preferred Reagent Cation for Negative Electron Transfer Dissociation. J Am Soc Mass Spectrom 28:1324-1332
Leach 3rd, Franklin E; Riley, Nicholas M; Westphall, Michael S et al. (2017) Negative Electron Transfer Dissociation Sequencing of Increasingly Sulfated Glycosaminoglycan Oligosaccharides on an Orbitrap Mass Spectrometer. J Am Soc Mass Spectrom 28:1844-1854
Riley, Nicholas M; Hebert, Alexander S; D├╝rnberger, Gerhard et al. (2017) Phosphoproteomics with Activated Ion Electron Transfer Dissociation. Anal Chem 89:6367-6376
Riley, Nicholas M; Westphall, Michael S; Coon, Joshua J (2017) Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation. J Am Soc Mass Spectrom :