Partition and Quantification of the Human Sera N-Glycome Utility for Disease Biomarker Discovery

Human glycoproteins are promising targets for disease research because they play an important role in many biological processes, including immune defense, cell growth, and cell-cell adhesion. Research shows that glycans on glycoproteins are active participants in protein stability, cell-cell interactions, and development. Moreover protein glycosylation in human serum is known to change with disease, pregnancy, age, and inflammation. However, studying protein glycosylation of whole serum is complicated due to the large number and broad dynamic range of serum glycoproteins. As a result glycomic profiling of human serum is complicated because of the numerous glycoproteins present in serum that vary in concentrations and extent of glycosylation. Lower abundant serum glycoprotein glycans are oftentimes missed in glycoproteomic analyses due to the glycans from more abundant glycoproteins overwhelming the glycan profile and precluding the observation of glycans from lower abundance glycoproteins. In addition to partitioning the human glycoproteome, the diversity in types, structure, monosaccharide composition, and abundances glycans present another analytical challenge.;Therefore, identifying the numerous glycoforms, including isomers, can help to elucidate the biological function(s) of particular glycans. To study the variations in glycosylation, establishment of analytical tools to address the depth of the human serum glycome complexity are necessary. Here methods to assess the diversity of the N-linked oligosaccharides released from human serum have been developed using affinity chromatography and mass spectrometry. N-Linked oligosaccharides were profiled using on-line nano-liquid chromatography (nanoLC) with high resolution time-of-flight mass spectrometry and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR MS). A method to then quantitate the N-glycans from immunoglobulin G, IgG, from human serum was developed to monitor the changes in N-glycosylation with lung cancer. Results indicate that the N-Linked glycans from IgG all decreased with lung cancer. Lastly, an alternative method to partition human serum using heparin affinity chromatography to isolate the proteins and N-glycans was developed. The proteins and the N-glycans in each fraction were studied. The heparin column partition showed an enhancement in identifying specific proteins and N-glycans.