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. 2023 Feb;120(2):491-502.
doi: 10.1002/bit.28280. Epub 2022 Nov 19.

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

Jelena Šimunović  1 , Jernej Gašperšič  2 , Urh Černigoj  2 , Jana Vidič  2 , Aleš Štrancar  2 , Mislav Novokmet  1 , Genadij Razdorov  1 , Marija Pezer  1 , Gordan Lauc  1   3 , Irena Trbojević-Akmačić  1
Affiliations

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

Jelena Šimunović et al. Biotechnol Bioeng. 2023 Feb.

Abstract

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.

Keywords: N-glycosylation; haptoglobin; high-throughput; immunoaffinity enrichment; monoliths.

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References

REFERENCES

    1. Alayash, A. I. (2011). Haptoglobin: Old protein with new functions. Clinica Chimica Acta, 412(7-8), 493-498.
    1. Brudar, S., Černigoj, U., Podgornik, H., Kržan, M., & Prislan, I. (2017). Use of differential scanning calorimetry and immunoaffinity chromatography to identify disease induced changes in human blood plasma proteome. Acta Chimica Slovenica, 64(3), 564-570.
    1. Buettner-Janusch, J. (1970). Evolution of serum protein polymorphisms. Annual Review of Genetics, 4(1), 47-68.
    1. Černigoj, U., Gašperšič, J., Fichtenbaum, A., Lendero Krajnc, N., Vidič, J., Mitulović, G., & Štrancar, A. (2016). Titanium dioxide nanoparticle coating of polymethacrylate-based chromatographic monoliths for phosphopetides enrichment. Analytica Chimica Acta, 942, 146-154.
    1. Černigoj, U., Vidic, U., Nemec, B., Gašperšič, J., Vidič, J., Lendero Krajnc, N., Štrancar, A., & Podgornik, A. (2016). Characterization of methacrylate chromatographic monoliths bearing affinity ligands. Journal of Chromatography A, 1464, 72-78.

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