Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

Abstract

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 nanograms. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 μL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3 linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1–3)-linked galactose in glycans

“…This has recently been demonstrated with a microarray that combined 2 neuraminidases and 7 lectins to provide information about both the structural motifs present in glycoproteins and the sialic acid linkage. [9]…”

Mining High-Complexity Motifs in Glycans: A New Language To Uncover the Fine Specificities of Lectins and Glycosidases

“…The new language ( Tables 1 and S-2 ) is expanded from a syntax introduced earlier. 22 We sought to develop a system that is flexible enough to describe any binding determinant, yet human-readable and easy to use. We also wanted notation that is immediately recognizable by those familiar with glycobiology and explicit definitions rather than common terms such as “Lewis X”.…”

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

“…Further, one could apply lectin profiling after rounds of exoglycosidase cleavage in order to obtain additional information about the structures. Lectin probing in the presence or absence of glycosidase digestion has been used in affinityelectrophoresis, lectin-blotting, and histochemical analysis (33)(34)(35)(36), and we have extended the approach to a micro-scale format in combination with algorithms for the automated interpretation of the data (37,38). We proposed that this method could provide complementary information to mass-spectrometry and be practical for researchers who are not experts in glycobiology.…”

“…Glycosidases are standard tools in glycan sequencing by chromatography and electrophoresis, and they are increasingly used in mass-spectrometry experiments. They also can be used to uncover motifs to be probed by lectins (37,38,86). In many cases, only basic information is known about the specificity of a glycosidase.…”