Signal amplification by glyco-qPCR for ultrasensitive detection of carbohydrates: applications in glycobiology.

The ultrasensitive determination of the carbohydrate components of glycoconjugates is challenging because of their low concentrations in complex biological mixtures and the chemical complexity of glycan chains. A major limitation in the field of glycobiology has been a lack of requisite analytical platforms for biologically active, therapeutic carbohydrates (e.g., glycosaminoglycans (GAGs) and sialylated glycans, such as N-glycans) and glycoconjugates (e.g., proteoglycans (PGs), glycoproteins (GPs), and glycolipids). Unlike nucleic acids, carbohydrates cannot be detected by amplification methods, and in contrast to proteins, there are few carbohydrate-specific antibodies. Moreover, carbohydrates have no natural chromophores or fluorophores, and often show poor ionization efficiency in mass spectrometry (MS). Methods for detecting carbohydrates in subfemtomole levels would greatly facilitate glycomics studies 5] on cell surface glycans, and improve our understanding of the biological roles of carbohydrates, protein glycosylation, and carbohydrate–protein interactions involved in the critical biological processes. 7, 8] We report a novel glyco-quantitative polymerase chain reaction (Glyco-qPCR) assay platform (Scheme 1) that allows the ultrasensitive detection and quantification of glycans in biological samples. We used chondroitin sulfate (CS) and sialylated N-glycans as model carbohydrates to demonstrate Glyco-qPCR. The CS GAG is a representative O-linked glycan side-chain of many biologically important PGs. Sialylated glycans are commonly found on GPs, PGs, and glycolipids. All target glycans in this study contain a free reducing end and a carboxy group, allowing the introduction of a double label of biotin and DNA. Ultrasensitive detection and quantification by GlycoqPCR relies on a high-affinity biotin–streptavidin interaction combined with qPCR amplification for molecular colocalization of glycan and DNA marker signal amplification of a target carbohydrate (Figure 1a). Because exponential amplification by PCR can permit the routine detection of even a single molecule of DNA, 9] we hypothesized that an appropriately designed assay might be similarly used to detect a single or small number of a carbohydrate–DNA conjugates. We also describe the application of Glyco-qPCR as a detection platform for the study of carbohydrate–protein interactions of critical importance in biology. 11] Glycan arrays, using fluorescent tags for high-throughput detection of carbohydrate–protein binding, often afford inconsistent data as a result of nonspecific interactions and variations in glycan ligand density and orientation on different array platforms. Glyco-qPCR might offer an ultrasensitive alternative to glycan arrays. CS GAG is a polydisperse, microhetereogenous mixture of polysaccharides that complicates its direct analysis. Thus, Scheme 1. Schematic representation of Glyco-qPCR. Target carbohydrates (GAGs) from biological samples can be conjugated with different DNA markers, followed by removing unreacted DNA and detecting the corresponding GAG–DNA conjugates with amplified signals (qPCR).