Combination of Nonspecific Proteolysis, Tandem Mass Spectrometry and Bioinformatics for Extensive Site-specific Glycosylation Analysis in Protein Mixtures

Proteins are a unique class of biological molecules known to perform a wide range of important functions. Amongst the numerous post-translational modifications (PTMs) that decorate proteins, glycosylation is one of the most common and diverse affecting about 70% of human proteins. Glycosylation is known to influence proper protein folding, increase protein stability and enhance cell-cell adhesion. These and many other functions make glycoproteins an important and interesting analyte to study. Protein glycosylation has been largely ignored in numerous traditional proteomic and glycomic experiments. This is partly due to the diverse and complex manner by which glycans particularly decorate proteins and the lack of effective analytical techniques available for comprehensively characterizing protein glycosylation. This dissertation addresses the current limitations of protein glycosylation analysis via strictly proteomic and glycomic strategies and it provides a solution. The effectiveness of the use of nonspecific proteolysis combined with mass spectrometric analysis towards achieving site-specific glycosylation in single purified proteins and in complex protein mixtures is described.;Chapter one 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 protein glycosylation. Chapter two presents a method for the enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry via matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). Negative ion mode is not commonly used in MS analyses, but appears well suited for glycopeptides.;An approach for the simultaneous and extensive site-specific N- and O-glycosylation analysis in a simple protein mixture is presented in chapter three. Here, immobilized pronase is shown to significantly reduce a mixture of N- and O-glycoproteins to glycopeptides, which are then investigated by nano-flow liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer (nano-LC/Q-TOF MS) and identified with an in-house data analysis software. This approach is shown to provide glycopeptide footprints that identify sites of protein glycosylation in a robust and sensitive manner. A targeted investigation of N- and O-glycosylation in complex protein mixtures via novel in-gel nonspecific proteolysis and analysis of the resulting N- and O-glycopeptides by nano-LC-MS/MS is described in chapter four. Finally, chapter five details a glycomic study involving the comparison of the human and bovine milk N-glycome via peptide N-glycosidase F (PNGase F) digestion and nano-LC-MS/MS analysis of the purified N-glycans.