In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS

Sialic acid Neu5Gc, a non-human glycan, is recognized as a new harmful substance that can cause vascular disease and cancer. Humans are unable to synthesize Neu5Gc due to a genetic defect that converts Neu5Ac to Neu5Gc, but Neu5Gc is often observed in human biological samples. Therefore, the demand for accurately measuring the amount of Neu5Gc present in human blood or tissues is rapidly increasing, but there is still no method to reliably quantify trace amounts of a non-human sugar. In particular, selective isolation and detection of Neu5Gc from human serum is analytically challenging due to the presence of excess sialic acid Neu5Ac, which has physicochemical properties very similar to Neu5Gc. Herein, we developed the label-free approach based on ZIC-HILIC/MRM-MS that can enrich sialic acids released from human serum and simultaneously monitor Neu5Ac and Neu5Gc. The combination of complete separation of Neu5Gc from abundant Neu5Ac by hydrophilic and electrostatic interactions with selective monitoring of structure-specific cross-ring cleavage ions generated by negative CID-MS/MS was remarkably effective for quantification of Neu5Ac and Neu5Gc at the femtomole level. Indeed, we were able to successfully determine the absolute quantitation of Neu5Gc from 30 healthy donors in the range of 3.336 +/- 1.252 pg/mu L (mean +/- SD), 10,000 times lower than Neu5Ac. In particular, analysis of sialic acids in protein-free serum revealed that both Neu5Ac and Neu5G are mostly bound to proteins and/or lipids, but not in free form. In addition, the correlation between expression level of Neu5Gc and biological factors such as BMI, age, and sex was investigated. This method can be widely used in studies requiring sialic acid-related measurements such as disease diagnosis or prediction of immunogenicity in biopharmaceuticals as it is both fast and highly sensitive.