Multi-Site Quantitative Benchmarking of Neu5Gc-Bearing N-Glycans Using Matrix-Matched LC-MS calibration

N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection in mass-spectrometric studies has remained elusive, but its low abundance has led to inconsistent detection in studies of human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC-MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC-MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC-MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, under the DMB-LC-FLR conditions and sample loading evaluated here, dynamic range was limited to two orders of magnitude, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that the DMB-LC-FLR assay's specificity and sensitivity were insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC-MS to improve the measurement of Neu5Gc in clinical samples.