Sialic acids (Sias) are glycan epitopes with critical roles in human biology and disease. Despite the fact that Sias are so widely and profoundly important across biology, not all Sias have received equal attention: short α2,8-linked Sia chains known as oligoSia are notably understudied. Our knowledge of oligoSia has lagged far behind that of other Sias in large part due to a lack of suitable methods with which to detect and characterize it. Unlike polySia, there is no commercially available antibody or lectin that recognizes oligoSia, making its detection challenging and isolation prohibitive.
To overcome these issues and shed light on the mystery that is oligoSia, we have developed an unbiased approach for the identification of novel oligosialylated proteins. We demonstrate the use of a chemical biology approach for the detection and quantification of oligoSia and identification of novel oligosialylated proteins, coined STaBL (sialyltransferase-associated biotin labelling). This strategy leverages a bacterial polysialyltransferase enzyme (1) with strict substrate specificity for α2,8-linked Sias to transfer a biotin-tagged sialic acid derivative. Resulting proteins were purified and analyzed by mass spectrometry. Dozens of novel oligosialylated proteins in serum and red blood cells were identified, including several minor blood group antigens.
The use of STaBL as a novel method for characterization of α2,8-linked Sias, including oligoSia and oligosialylated proteins, presents a solution to overcome the long-standing challenges involved in studying this glycan. The method is straightforward and compatible with common analytical techniques, including blotting, immunoprecipitation, ELISA, flow cytometry, and mass spectrometry. This method is therefore highly versatile and could be employed in a variety of contexts to advance our understanding of the roles of oligoSia in health and disease. Our initial use of STaBL revealed insights into how α2,8-linked Sias on red blood cells were impacted by biological age and cell storage conditions, with profound implications for transfusion medicine. These initial findings provided clues to understanding the RBC storage lesion and preventing adverse events associated with RBC transfusion, while the broader application of our method will accelerate understanding of the role of α2,8-linked Sias in health and in diverse diseases.