a-quantitative-glycomics-and-proteomics-combined-purification-strategy

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

North Carolina State University

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.

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

An early-stage, population-wide biomarker for ovarian cancer (OVC) is essential to reverse its high mortality rate. Aberrant glycosylation by OVC has been reported, but studies have yet to identify an N-glycan with sufficiently high specificity. We curated a human biorepository of 82 case-control plasma samples, with 27%, 12%, 46%, and 15% falling across stages I-IV, respectively. For relative quantitation, glycans were analyzed by the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) strategy for enhanced electrospray ionization, MS/MS analysis. Sixty-three glycan cancer burden ratios (GBRs), defined as the log10 ratio of the case-control extracted ion chromatogram abundances, were calculated above the limit of detection. The final GBR models, built using stepwise forward regression, included three significant terms: OVC stage, normalized mean GBR, and tag chemical purity; glycan class, fucosylation, or sialylation were not significant variables. After Bonferroni correction, seven N-glycans were identified as significant (p < 0.05), and after false discovery rate correction, an additional four glycans were determined to be significant (p < 0.05), with one borderline (p = 0.05). For all N-glycans, the vectors of the effects from stages II-IV were sequentially reversed, suggesting potential biological changes in OVC morphology or in host response.

Article	Year
Definitive Screening Design Optimization of Mass Spectrometry Parameters for Sensitive Comparison of Filter and Solid Phase Extraction Purified, INLIGHT Plasma N-Glycans. Analytical chemistry\| PubMed ID: 26086806	2015
Relative Quantification and Higher-Order Modeling of the Plasma Glycan Cancer Burden Ratio in Ovarian Cancer Case-Control Samples. Journal of proteome research\| PubMed ID: 26347193	2015

Elizabeth S. Hecht

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