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.