Mass spectrometry-based shotgun glycomics (MS-SG), which utilizes catch-and-release electrospray ionization (CaR-ESI)-MS screening, is a powerful approach for mapping the glycan interactome of glycan-binding proteins (GBPs) and is poised to play a dominant role in the area of functional glycomics. However, the affinities of ligands identified from CaR-ESI-MS screening cannot be directly established due to the challenge in determining glycan concentrations in natural libraries. In this work, we explore the approach of combining screening data with relative glycan concentrations established by liquid chromatography (LC) analysis of the fluorophore-labeled library to produce affinity rankings and compare the influence of three different labeling strategies (2-AA, 2-AB and proA). Differences, in some cases dramatic, were found for affinity rankings obtained from the libraries labeled with the three different fluorophores, and those produced using the combined unlabeled/labeled glycan library approach. The origin of these differences can be explained by the combined differential effects of glycan labeling efficiencies, solid phase extraction recovery, glycan-GBP affinities, and release efficiencies in CaR-ESI-MS. Overall, the 2-AB(CaR-ESI-MS)/2-AB(LC) combination produces the most reliable description of the glycan binding specificities for the panel of GBPs investigated. With the goal of eliminating the deleterious effects of labeling in MS-SG, efforts are now being directed towards establishing a label-free MS approach for the quantitative screening of natural glycan libraries. Preliminary data obtained using two different experimental strategies will also be presented.

Advances in mass spectrometry-based glycan library screening

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ACS Fall 2022

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