Integrating Chemoselective Labeling and Laser-Cleavable Mass Tagging for Determination of Sialic Acids in Glycoconjugates.

Sialic acids are the terminal units of glycans in glycoproteins or glycolipids. The determination of sialic acids in glycoconjugates is crucial since they regulate essential biological functions and have a significant nutritional value. To achieve a specific and high-throughput in situ determination of sialic acids in glycoconjugates, a laser-desorption/ionization mass spectrometry (LDI-MS)-based strategy is reported by integrating chemoselective labeling and laser-cleavable mass tagging. 1-Pyrenebutyric hydrazide (PBH), a commercially available reagent that contains a pyrene moiety and a hydrazide group, has been developed as a novel laser-cleavable mass tag. For chemoselective labeling, an aldehyde group is introduced to the polyhydroxy side chain of sialic acids through mild periodate oxidation and then reacted with PBH, achieving the in situ determination of sialic acids. The quantitative determination of sialic acids in the range of 5-1000 μM (R = 0.99984) was achieved using an internal standard method. Thus, a specific, quantitative, and high-throughput method was developed for the in situ determination of sialic acids in glycoconjugates. Finally, this method has been successfully used to quantify the sialic acid content in EBN proteins and glycoprotein biopharmaceuticals, showing its practical application in the quality control of nutritional and therapeutic glycoprotein products. Additionally, the pyrene moiety, when linked to other reactive groups, can also be utilized to analyze other biomolecules, offering a new route for the rational design of mass tags.