Semi‐high‐throughput isolation and N N ‐glycan analysis of human fibrinogen using monolithic supports bearing monoclonal anti‐human fibrinogen antibodies

摘要

Abstract Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N ‐glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti‐human fibrinogen antibodies in a single column as well as in multi‐well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi‐high‐throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N ‐glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies.