In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis.

Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan's lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separation or detection, and to enhance the sensitivity of the analysis. Released glycan's are usually derivatized using a fluorescent tag, which enables their optical detection in LC profiling. Some fluorescent labels can also promote ionization efficiency, thus facilitating MS detection. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycan's and the need to quantify these contributions to improve glycan analysis methods. In this paper we focused on negative MS mode as these methods are more informative than methods involving positive MS mode, allowing for a less ambiguous elucidation of detailed glycan structures. Additionally, traditional glycan labels in negative mode MS usually result with diminished sensitivity compared to positive mode, thus making selection of appropriate label even more important for successful high-throughput analysis. Therefore, eleven fluorescent labels of different chemo-physical properties were chosen to study the influence of label hydrophobicity and presence of a negative charge on glycan ionization in negative MS mode. N-glycans released from IgG sample were labeled with one of the eleven labels, purified with HILIC-SPE and analyzed with HILIC-UPLC-FLR-MS. To make evaluation of studied labels performance more objective, analysis was performed in two laboratories and at two mobile phase pH (4.4 and 7.4). Although there was a notable trend of more hydrophobic labels having bigger signal intensities in one laboratory, we observed no such trend in the other laboratory. The results show that MS parameters and intrinsic configuration of the spectrometer have even bigger effect on the final ESI response of the labeled-glycan ionization in negative MS mode that the labels themselves. With this in mind, further research and development of fluorophores that will be suitable for high-throughput glycan analysis in the negative MS mode are proposed.