Izvorni znanstveni članak

https://doi.org/10.5562/cca2815

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

Irena Trbojević-Akmačić ; Genos Glycoscience Research Laboratory, Hondlova 2/11, HR-10000 Zagreb, Croatia
Blaž Nemec ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Urška Vidic ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Suzana Malić ; Center for Proteomics, University of Rijeka, Faculty of Medicine, B. Branchetta 20, HR-51000 Rijeka, Croatia
Karmela Miklić ; Center for Proteomics, University of Rijeka, Faculty of Medicine, B. Branchetta 20, HR-51000 Rijeka, Croatia
Urh Černigoj ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Jana Vidič ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Nika Lendero Krajnc ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Aleš Štrancar ; BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia
Gordan Lauc ; Genos Glycoscience Research Laboratory, Hondlova 2/11, HR-10000 Zagreb, Croatia
Tihana Lenac Roviš ; Center for Proteomics, University of Rijeka, Faculty of Medicine, B. Branchetta 20, HR-51000 Rijeka, Croatia
Maja Pučić-Baković orcid.org/0000-0003-0866-623X ; Genos Glycoscience Research Laboratory, Hondlova 2/11, HR-10000 Zagreb, Croatia

Sažetak

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 high-throughput 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.

This work is licensed under a Creative Commons Attribution 4.0 International License.

Ključne riječi

high-throughput; immunoaffinity chromatography; monoliths; <i>N</i>-glycosylation; oriented antibody immobilization; transferrin

Hrčak ID:

166006

URI

https://hrcak.srce.hr/166006

Datum izdavanja:

21.6.2016.

Posjeta: 2.725 *