Attempts at glycan sequencing has introduced numerous strategies. Rarely, if ever, are such reports coupled with details that allow comprehensive evaluation. Carbohydrate sequencing has become a playground where anything goes in publications. Lost in the maize of high resolution and high energy MS operation has been low energy disassembly provided by the first triple quadrupoles and carried further with the ion trap. Here neutral gas activated carbohydrate ions are disassociated in a step-wise, structurally dependent manner to yield regio-specific linkage fragments. Importantly, such ions can be captured, documented and shared, retaining all aspects of structure that reversibly provides sequence information. Extensive studies for over two decades have shown the fidelity of disassembly with a large cross selection of synthetic and natural carbohydrate samples and identical results have also been observed in both linear and quadrupolar ion traps, supporting the concept that following low energy activation, disassembly is an independent physiochemical process controlled only by its inherent structure. Moreover, three additional facts complement this approach as an effective sequence technology; 1.) a loss of native background ions with early MS<b><sup>n</sup></b> steps, allows commensurate sample ion amplification; 2.) double and triple charging significantly extends mass range; and, 3.) Incrementing steps of disassembly occur at higher energies, that must be shared with smaller oscillator number, a feature that provides greater structural detail in core areas of stability. This latter feature is predicted by the quasi-equilibrium theory, (RRKM). With support from these basic physiochemical facts, we turned our attention to challenging stereo isomers, as known in the equatorial-axial pyran isomers, (the C4 Gal/Glc, GlcN/GalN and the C2 Glc/Man monomers). Synthetic regio-specific disaccharide samples that included these specific stereoisomers did produce differential spectra upon disassembly, completing a spatial regio-specific analysis. Since synthesis is revered as a proof of structure, so in a reverse sense, disassembly, based on losses, eliminations and rearranged products, also provides product ions that document precursor structure. An accumulation of such spectra in a searchable spectral data file would greatly facilitate collaborative studies, a component long missing in glycosylation research.

Comprehensive glycan sequencing with documented results

256th National Meeting (2018)

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