Abstract
We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media – nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.
Highlights
HeLa is the most commonly analyzed immortalized cell line in biomedical research[1]
Initial proteomics analysis of the commercial, widely used HeLa standard revealed that the sample contains a large amount of bovine serum proteins, most likely from fetal bovine serum (FBS), present in the cell culture media
To estimate the relative abundance of individual glycoproteins, label-free quantitation was performed on the un-enriched HeLa cell lysate
Summary
HeLa is the most commonly analyzed immortalized cell line in biomedical research[1]. It is a human cervical adenocarcinoma cell line expressing over 10 000 proteins[2]. Understanding N-glycosylation patterns is important, as almost all cell surface proteins are glycosylated and it is assumed that without these glycoproteins the cells cannot survive[12]. It is not yet completely clear how the N-glycans influence the function or the fate of the N-glycoproteins. N-glycosylation sites of HeLa cellular proteins are fairly well referred using hydrazide enrichment followed by nanoLC-MS/MS analysis[11]. Using this method 268 unique N-glycosylation sites corresponding to 106 glycoproteins have been identified so far[11]. Aberrant glycosylation is considered a hallmark of cancer since glycosylation is involved in malignant properties of cancer cells[23,24]
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