Capillary electrophoresis–mass spectrometry for direct structural identification of serum N-glycans

Abstract

Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Snyder et al. developed a sheathless CE‐MS method, identifying 77 potential N ‐glycan structures in human serum .…”

Capillary Electrophoresis Separations of Glycans

“… 98 , 101 Alternatively, the electrospray ionization techniques occur almost exclusively in-line with the CE separation. 91 , 102 − 104 Sheathless interfaces that introduce ion flow to complete the electrical circuit are fragile, but as shown in Figure 9 , have been engineered to be easier for the user to manipulate and operate. 106 Glycan structure can be elucidated from electrospray ionization without fragmentation by employing mass deconvolution, 98 a combination of exoglycosidases and exact mass.…”

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis

“…Because of such difficulties, though in the last few years important developments of CE ESI MS approaches, some even introducing chips and automation or combining the on‐line separation with top‐down fragmentation for protein characterization and metabolomics were reported, much less was achieved in innovations dedicated to glycoconjugate analysis. Still a number of original publications presenting excellent protocols and several reviews highlighting in details the advancements of the method for general glycomics are available .…”