PROJECT SUMMARY Glycans have several distinct properties that make them potential disease biomarkers. Firstly, their location on cell surfaces makes them the first point of contact for cellular interactions, and thus they are crucial in the control of normal metabolic processes. Secondly, cell surface molecules are strategically exposed for surveillance by the immune system, where they may serve as immune recognition elements. Thirdly, specific glycan structures that are not present, or are in low amounts in normal states, proliferate in disease states, such as cancer. For example, variation in glycan sialylation is a marker for diseases, such as rheumatoid arthritis and prostate cancer, however, to fully exploit sialic acid as a disease marker requires the ability to detect it in each of its linkage contexts. At present multiple lectins are employed in serial lectin affinity chromatography to enrich sialylated glycans for glycomic analysis, which because of specificity issues associated with the lectins risks capturing irrelevant non-sialylated components. To overcome the limitations of existing lectins, we have used structurally-guided mutagenesis, to convert the carbohydrate-processing enzyme NanB (from S. pneumoniae) into a high-affinity reagent (Sia-3S1) specific for detecting 3'-sialylated glycans. Sia-3S1 shows significant advantages over existing reagents in chromatographic applications that are commonly used in disease marker detection. Such engineered proteins are referred to as ?Lectenz? because they have lectin-like properties, but retain the specificity of the endogenous enzymes. Lectenz have several advantages over either lectins or antibodies, including precise definition of specificity, tuneable affinity, and ease of production. Few reagents exist that can be used to enrich a sample for unique glycan modifications. Specific Lectenz such as Sia-3S1 directly address the needs of glyco-biomarker detection and mass spectrometry-based glycomics/proteomics analysis by enabling or improving sample enrichment. It could also be employed in histological studies, or Western blots, etc. In this application we illustrate that the development of Sia-3S1 has passed the proto-type stage, and demonstrate the scientific and technical merit and feasibility of advancing this technology into a highly desirable reagent and commercializable suite of tools.