MSACL 2019 EU Abstract

Analysis of the Site-Specific N-Glycosylation of HeLa Cell Lysate

Simon Sugár (1), András Ács (1), Ágnes Gömöry (1), Gábor Tóth (1,2), Károly Vékey (1), László Drahos (1), Lilla Turiák (1)
(1) MS Proteomics Research Group, Hungarian Academy of Sciences, Research Centre for Natural Sciences, Budapest, Hungary (2) Budapest University of Technology and Economics, Faculty of Chemical Technology and Biotechnology, Budapest, Hungary

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Simon Sugár (Presenter) Hungarian Academy of Sciences >> POSTER (PDF)

Presenter Bio: Simon Sugár the presenter of the poster got his BSc in chemistry at Eötvös Loránd University in Budpaest, then his MSc in chemical engineering at the Budapest University of Technology and Economics where he specialized in analytical chemistry. He has been the member of the MS Proteomics Research Group at the Hungarian Academy of Sciences Research Centre for Natural Sciences since 2016. He is currently researching site-specific N-glycosylation.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: The analysis of glycoproteins in biological samples is a challenging task. Routine glycoproteomics approaches involve sample enrichment, proteolytic digestion followed by de-glycosylation, the analysis of components with HPLC-MS/MS and data assessment via database search. However, during de-glycosylation the site-specificity of the information is lost, and during DDA MS/MS analyses many minor components are overlooked.
The use of HeLa cells in cancer research is prevalent and it is also the most commonly used mass spectrometry reference standard for proteomics. Although they have been extensively studied, there is very little information available on protein glycosylation in HeLa cells.

OBJECTIVES: Our aim was the determination of the site-specific N-glycosylation pattern of the proteins in a commercially available HeLa cell lysate.

METHODS: Here we introduce a novel workflow, which combines a glycopeptide enrichment and LC-MS analysis; the latter involving structural analysis using data dependent and energy resolved MS/MS, and quantitative analysis using MS1. This provides both excellent sensitivity, and reliable structure assignments. Our focus was not on maximizing the quantity of the identified glycopeptides, but rather on enhancing the reliability of the results. For this reason, we incorporated a multi-step validation protocol into the data evaluation workflow.

RESULTS: We identified over 40 human glycoproteins, with 69 glycosylation sites, 28 different glycans and altogether 178 glycopeptides with high confidence. Among these we have identified both core- and antenna-fucosylated glycoproteins, complex, high-mannose and hybrid N-glycosylated structures using special low-energy MS/MS experiments. We have also discovered very high amounts of bovine serum protein contaminants in the HeLa cell lysate. This suggests that previous studies – especially released glycan analyses – on HeLa glycosylation may have been biased, as the glycosylation pattern of cellular and serum proteins is different. This presumption is strongly supported by the results of the comparison of our data to a recent study on HeLa glycosylation based on released glycan analysis. [1]

CONCLUSION: The first detailed, protein- and site-specific N-glycosylation analysis of the HeLa cell line is presented. Our data also suggests that HeLa glycosylation should only be studied in a site-specific manner to avoid bias resulting from the presence of the culture media.

ACKNOWLEDGEMENTS, REFERENCES: LT and KV are grateful for funding from the National Research Development and Innovation Office (NKFIH PD-121187 and NKFIH K-119459). LT is grateful for support from the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.
[1]: Gao, Wenjie, et al. "A facile method for cellular N-glycomic profiling by matrix-assisted laser desorption/ionization mass spectrometry." RSC Advances 7.57 (2017): 35687-35693.