High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Autor: Šimunović J; Genos Glycoscience Research Laboratory, Zagreb, Croatia., Gašperšič J; Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia., Černigoj U; Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia., Vidič J; Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia., Štrancar A; Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia., Novokmet M; Genos Glycoscience Research Laboratory, Zagreb, Croatia., Razdorov G; Genos Glycoscience Research Laboratory, Zagreb, Croatia., Pezer M; Genos Glycoscience Research Laboratory, Zagreb, Croatia., Lauc G; Genos Glycoscience Research Laboratory, Zagreb, Croatia.; Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia., Trbojević-Akmačić I; Genos Glycoscience Research Laboratory, Zagreb, Croatia.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2023 Feb; Vol. 120 (2), pp. 491-502. Date of Electronic Publication: 2022 Nov 19.
DOI: 10.1002/bit.28280
Abstrakt: Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.
(© 2022 Wiley Periodicals LLC.)
Databáze: MEDLINE
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