Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

Abstract

Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf) glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific highthroughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC) analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility.

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

“…The dynamic binding capacity (DBC) and the dynamic binding capacity at 50% breakthrough (DBC 50 ) were determined for the CIMac pA‐@FIB and CIMac‐@FIB columns, as well as for the CIMac‐@FIB 8‐well plate as previously described . In short, FIB dissolved in PBS was loaded on the column or added to the plate wells until the point of breakthrough.…”

“…N ‐glycans were prepared and analysed by UPLC as previously described . In short, dried FIB eluates were deglycosylated with PNGase F after protein denaturation with SDS at 65°C for 10 min.…”

“…Purified FIB N ‐glycans were analysed by HILIC‐UPLC under control of Empower 3 software, build 3471 (Waters) as previously described . N ‐glycan chromatographic peaks (GP1‐GP24) were expressed as percentage of total integrated area (% Area).…”

“…The use of polymethacrylate monoliths as a chromatographic support enables flowrate‐independent binding capacity and resolution for large biomolecules due to the convective nature of the flow . Chromatographic monoliths have already proven to be an appropriate support for immunoaffinity chromatography , but to our knowledge there is only one report combining immunoaffinity sorbents with chromatographic monoliths in a 96‐well format .…”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…Another study described rapid purification of erythropoietin from biological samples on 6 μL disposable monoliths containing immobilized anti‐erythropoietin Ab . Purification of transferrin from human plasma was performed in HTP fashion with 96‐well plate format of monolithic support with anti‐transferrin monoclonal antibody immobilized over its glycan component. A 200 μL monolith per well was employed and 300 μg of transferrin was obtained, that was enough for further HTP profiling of its N‐glycans.…”