The analysis of glycopeptides spans many different areas of research, from investigators probing subtle changes of glycosylation due to pathological conditions to scientists characterizing biopharmaceutical products. However, glycopeptides present several unique analytical challenges in their overall characterization, including suppressed mass spectral (MS) ionization efficiencies when they are present as lower abundance components of complex mixtures. One approach to improve their MS performance is to first employ a state-of-the-art ultraperformance liquid chromatographic (UPLC) separation technique. Such separations are routinely performed as part of peptide mapping experiments as part of the overall characterization of biotherapeutic products. These experiments most frequently rely on reversed-phase chromatographic methods which separate analytes based on their hydrophobic characteristics. Unfortunately, the effectiveness of this technique to efficiently resolve the glycoforms of a single peptide backbone is limited and significant coelution analytes occurs. Alternatively, however, hydrophilic interaction liquid chromatography (HILIC) is a technique that is widely used for efficient separations of both small and large molecules with hydrophilic characteristics. Using optimized mobile-phase conditions for an amide-functionalized UPLC HILIC column, the glycan becomes the critical component driving the separation. Using this mode of chromatography, different glycoforms of a single peptide backbone are well resolved. Thus, HILIC allows the glycan microheterogeneity of a specific site of glycosylation to be accurately mapped and quantitated if an optical detection technique, like tunable UV, is employed. In this presentation, we will discuss optimizing mobile phase conditions to balance chromatographic performance and MS sensitivity. We will also present glycopeptide mapping results for standard and biotherapeutic proteins which demonstrates the ability of UPLC/HILIC/MS to characterize specific sites of glycosylation.

Advancing glycopeptide characterization using high sensitivity HILIC-MS methods

253rd National Meeting (2017)

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