In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry

Abstract

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

“…Efficient isomeric separation of oligosaccharides and glycans by HPAE chromatography under alkaline conditions has been reported by some researchers [109,113–115]. For example, Szabo et al.…”

“…For example, Szabo et al. analyzed native N‐ glycans using a CarboPac® PA200 column, showing that HPAE has an excellent resolution between positional and linkage isomers of charged sialylated glycans [113]. Later, the researchers compared HPAE‐MS and LC‐MS by analyzing 2‐AB labeled N‐ glycans of recombinant human erythropoietin [115].…”

Clinical application of quantitative glycomics

“…Benefiting from the compatibility with MS and high separation resolution, LC-MS is widely used in clinical glycomic profiling [29]. A variety of LC techniques have been employed to characterize glycosylation patterns, including hydrophilic interaction liquid chromatography (HILIC) [36-43], reverse phase liquid chromatography (RPLC) [44-47], high-pH anion exchange chromatography (HPAEC) [48,49] and liquid chromatography using porous graphitic carbon (PGC) [15,45,50-55] have been previously demonstrated and elaborated reviewed [56,57].…”

Translating N‐Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer

“…CAD offers a better performance than other detectors, such as refractive index (RI), low wavelength UV, and evaporative light scattering (ELS). Alternatively, high‐performance anion exchange (HPAE) coupled with pulsed amperometric detection (PAD) has also emerged to quantitatively analyze glycans cleaved from glycoproteins . HPAE‐PAD is capable of measuring glycans in the low picomole range.…”