Definitive Screening Design Optimization of Mass Spectrometry Parameters for Sensitive Comparison of Filter and Solid Phase Extraction Purified, INLIGHT Plasma N-Glycans.

High-throughput, quantitative processing of N-linked glycans would facilitate large-scale studies correlating the glycome with disease and open the field to basic and applied researchers. We sought to meet these goals by coupling filter-aided-N-glycan separation (FANGS) to the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) for analysis of plasma. A quantitative comparison of this method was conducted against solid phase extraction (SPE), a ubiquitous and trusted method for glycan purification. We demonstrate that FANGS-INLIGHT purification was not significantly different from SPE in terms of glycan abundances, variability, functional classes, or molecular weight distributions. Furthermore, to increase the depth of glycome coverage, we executed a definitive screening design of experiments (DOE) to optimize the MS parameters for glycan analyses. We optimized MS parameters across five N-glycan responses using a standard glycan mixture, translated these to plasma and achieved up to a 3-fold increase in ion abundances.