The sweet spot: The combination of nonspecific proteolysis and mass spectrometry for glycoproteomic analysis.

摘要

Proteins have a wide range of biological functions; their biological activities have also been observed to be dependent upon their post translational modifications (PTMs). Glycosylation is one of the most common PTMs, and has been recognized to encourage proper protein folding, increase protein stability, and enhance cell-cell adhesion. These and other benefits of glycosylation allow glycoproteins to recognize functions and activities that would otherwise be prohibited. Even though glycosylation has a significant influence on protein function it has been largely ignored during protein characterization due to the shortcomings of the available analytical techniques for glycoprotein analysis. This dissertation addresses the current pitfalls of glycoproteomics and demonstrates the effectiveness of non-specific proteolysis combined with mass spectrometric analysis for the characterization of site-specific protein glycosylation.;The first chapter provides an introduction to glycoproteomics including a summary of common methods used to investigate protein glycosylation. Chapter one concludes with an overview of mass spectrometry and recent advancements that have permitted its use for the characterization of biomolecules. Chapter two presents a method for the rapid characterization and quantitation of Fructooligosaccharides (FOS) via matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). FOS is a commonly utilized food additive with proposed prebiotic benefits. The novel method will provide a platform for the investigation of this potential prebiotic in a quantitative, sensitive, and rapid manner.;A comprehensive approach for profiling protein glycosylation is presented in chapter three. Immobilized pronase is shown to reproducibly reduce glycoproteins to glycopeptides, which are then investigated by FT-ICR MS and identified with data analysis software. This approach is shown to provide glycopeptide footprints that identify sites of protein glycosylation in high throughput, robust, and sensitive manner. The dissociation behaviors of N-linked (chapter four) and O-linked (chapter five) glycopeptides are then investigated to allow for the complete characterization of protein glycosylation. Glycopeptide dissociation is shown to be greatly dependent upon charge carrier and the core structure of the attached oligosaccharides. The combination of glycopeptide footprinting and tandem mass spectrometry is utilized to characterize many glycoproteins indicating that the past limitations of glycoproteomics are overcome with this method.