Analysis of glycoprotein polypeptide sites of carbohydrate attachment and the structures of the glycans present on these proteins requires the combination of high resolution chromatographic separations and sophisticated mass spectrometry. Hydrophilic interaction liquid chromatography (HILIC) has great use in separations of glycopeptides and released glycans, exhibiting remarkable selectivity for the separations of glycoforms. The proposed effort is to markedly improve separations of glycopeptides and released glycans by HILIC, reducing the separation times from the current 2-3 hours per sample, to less than 30 minutes. The separations component of LC/MS analysis of glycoproteins can benefit from the design of more efficient stationary phase chromatographic materials, and from the use of conditions that allow direct and productive interfacing with mass spectrometers. We propose that recently developed superficially-porous silica microparticulate silica packing materials (Fused-Core(R) structures) can achieve these separation speeds, without loss of the required high resolution. The AMT Fused-Core materials have previously been shown to exhibit superior kinetic properties and column efficiencies for separations of small molecules, and recently, wider pore size packings have shown a similar benefit for larger peptides (c. 3-5 kDa). The current proposal uses Fused-Core technology with unique surface modifications to develop high performance HILIC materials specifically for separations of glycopeptides and glycans. Pore size and surface tailored HILIC materials will be synthesized and tested for HILIC operation, as well as to establish the fundamental relationships between surface properties, particle characteristics, and utility for glycan and glycopeptides separations. The new bonded phases will utilize organosilane reagents selected to withstand aggressive conditions of use (low pH and elevated temperatures), permitting stable and robust separations materials that can be used across a broad range of conditions. A reasonable estimate is that an improvement of 2-5 fold in separations times can be obtained, compared with current methods. The current proposal will further assess the benefits and real-world applications of these new separations materials by use in glycoprotein structural analysis. The target of this work is to produce robust materials that permit simple integration with online mass spectrometry analysis, effective for resolution of the complex mixtures that are typical of current glycoprotein identification and structural characterization procedures.
Protein modification by the addition of sugars (called glycosylation) is extremely common in nature, and this modification exhibits a strong effect on protein structure and on biological function. In certain diseases, most notably in cancer, glycosylation of proteins is altered in ways that are not fully understood, but which are sufficiently different to allow such modifications to be used for recognition of disease, and in some cases to stratify patients. The current proposal is to use new knowledge in materials science and chemistry to enable much faster and efficient ways to discover more, and higher quality glycosylation disease markers.
