High performance IT-MSn sequencing of glycans: Spatial resolution of ovalbumin isomers

This report outlines and applies a high performance sequencing technology to evaluate the glycome of a common model glycoprotein, ovalbumin. The targets were the N-linked glycans enzymatically released from the protein, the N-glycoproteome. These product glycans were reduced, methylated and directly infused into the MS using a chip-based nanoelectrospray with the ions structurally characterized by sequential disassembly. Ten major ions were selected for detailed analysis. Isomer topologies (glycan connectivity) were determined from ion pathways of disassembly. Linkage information was revealed by specific cross-ring cleavage fragments within smaller oligomers. Both connectivity and linkage features were assisted with described bioinformatic tools and details confirmed with a standards library of fragments. The number of isomeric structures found within these 10 parent ions were 37, more than double earlier reports, and setting a new goal for developing technology. In this non-chromatographic, high performance spatial approach, the focus has been patterned to be comprehensive, and stay within the bounds of a plausible high throughput strategy consistent with automation. Selective structures are described in the text to appraise readers of the general approach; a more comprehensive coverage has been included in supplemental material.

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► Non-chromatographic, high performance spatial approach to resolve the N-glycans of ovalbumin.
► IT-MSn disassembly with fragment ion library confirmation.
► Sequencing technology patterned to be comprehensive, and stay within the bounds of a plausible high throughput strategy consistent with automation.
► Considerations may offer some reprieve from this two-century-old problem of carbohydrate sequencing.