So You Discovered a Potential Glycan-Based Biomarker; Now What? We Developed a High-Throughput Method for Quantitative Clinical Glycan Biomarker Validation

Abstract

Glycomic-based approaches to discover potential biomarkers have shown great promise in their ability to distinguish between healthy and diseased individuals; these methods can identify when aberrant glycosylation is significant, but they cannot practically be adapted into widely implemented diagnostic assays because they are too complex, expensive, and low-throughput. We have developed a new strategy that addresses challenges associated with sample preparation, sample throughput, instrumentation needs, and data analysis to transfer the valuable knowledge provided by protein glycosylation into a clinical environment. Notably, the detection limits of the assay are in the single-digit picomole range. Proof of principle is demonstrated by quantifying the changes in the sialic acid content in fetuin. As the sialic acid content in proteins varies in a number of disease states, this example demonstrates the utility of the method for biomarker analysis. Furthermore, the developed method can be adapted to other biologically important saccharides, affording a broad array of quantitative glycomic analyses that are accessible in a high-throughput, plate-reader format. These studies enable glycomic-based biomarker discovery efforts to transition through the difficult landscape of developing a potential biomarker into a clinical assay.

Direct Glycan Analysis of Biological Samples and Intact Glycoproteins by Integrating Machine Learning-Driven Surface-Enhanced Raman Scattering and Boronic Acid Arrays

“…Therefore, the direct analysis of intact glycoproteins without tedious sample preparation is highly desired. For the proof-of-concept, we selected fetuin and asialofetuin as a model system . Bovine fetuin is known to contain three N -glycosylation sites and five O-glycosylation sites, while asialofetuin shares the same protein structure and glycosylation sites but lacks terminal sialic acids .…”

Direct Glycan Analysis of Biological Samples and Intact Glycoproteins by Integrating Machine Learning Driven- Surface-Enhanced Raman Scattering (SERS) and Boronic Acid Array

“…Therefore, direct analysis of intact glycoproteins without tedious sample preparation is highly desired. For the proof-of-concept, we selected fetuin and asialofetuin as a model system 43 . Bovine fetuin is known to contain three Nglycosylation sites and five O-glycosylation sites 44 , while asialofetuin shares the same protein structure and glycosylation sites but lacks terminal sialic acids 45 .…”

Glycosylation Alterations in Cancer Cells, Prognostic Value of Glycan Biomarkers and Their Potential as Novel Therapeutic Targets in Breast Cancer

“…Although potential glycan biomarkers are being rapidly discovered and their value is supported by a significant amount of evidence, these approaches face difficulties in being adapted into widely implemented diagnostic assays due to their complexity, price, and low throughput. A significant number of studies such as the one mentioned above, enable potential glycan-based biomarkers discoveries to develop into clinically useful assays [ 113 , 114 ]. Another issue with developing clinically practical biomarkers is the need to ensure that they meet specific requirements—sensitivity and specificity.…”