Revealing the Biological Attributes of N-Glycan Isomers in Breast Cancer Brain Metastasis Using Porous Graphitic Carbon (PGC) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract

Breast cancer is a leading cancer in women and is considered to be the second-most common metastatic cancer following lung cancer. An estimated 10–16% of breast cancer patients are suffering from brain metastasis, and the diagnostic cases of breast cancer brain metastasis are increasing. Nevertheless, the mechanisms behind this process are still unclear. Aberrant glycosylation has been proved to be related to many diseases and cancer metastasis. However, studies of N-glycan isomer function in breast cancer brain metastasis are limited. In this study, the expressions of N-glycan isomers derived from five breast cancer cell lines and one brain cancer cell line were investigated and compared to a brain-seeking cell line, 231BR, to acquire a better understanding of the role glycan isomers play in breast cancer brain metastasis. The high temperature nanoPGC-LC-MS/MS achieved an efficient isomeric separation and permitted the identification and quantitation of 144 isomers from 50 N-glycan compositions. There were significant expression alterations of these glycan isomers among the different breast cancer cell lines. The increase of total glycan abundance and sialylation level were observed to be associated with breast cancer invasion. With regard to individual isomers, the greatest number of sialylated isomers was observed along with significant expression alterations in 231BR, suggesting a relationship between glycan sialylation and breast cancer brain metastasis. Furthermore, the increase of the α2,6-sialylation level in 231BR likely contributes to the passage of breast cancer cells through the blood-brain barrier, thus facilitating breast cancer brain metastasis. Meanwhile, the upregulation of highly sialylated glycan isomers with α2,6-linked sialic acids were found to be associated with breast cancer metastasis. This investigation of glycan isomer expressions, especially the unique isomeric expression in brain-seeking cell line 231BR, provides new information toward understanding the potential roles glycan isomers play during breast cancer metastasis and more clues for a deeper insight of this bioprocess.

Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers

“…An alternative chromatographic mode to RP-LC in glycopeptide analysis is to use porous graphitized carbon (PGC). PGC has been successfully applied in the isomeric separation of different N -glycopeptides [ 13 , 14 ]. Another chromatographic option for glycopeptide separation is hydrophilic interaction liquid chromatography (HILIC) [ 15 ].…”

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

“…In this work, we demonstrated isomeric separation of permethylated O-glycans, free oligosaccharides, and GSL-glycans using a PGC-LC technique that has been previously shown to separate permethylated N-glycans [28][29][30][31]. Several separation techniques for O-glycans were used, including the utilization of ion mobility [34] and PGC-LC [34,35] for analysis of native O-glycans.…”

“…We have reported the efficient isomeric separation of N-glycans previously [28][29][30][31]. Here, we demonstrate the separation of smaller glycans such as O-glycans, free oligosaccharides, and glycosphingolipid-glycans (GSL-glycans) using permethylation and PGC-LC-MS to resolve isomeric glycans at a high sensitivity.…”

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

“…Aberrant glycosylation has proved to be related to the progression of several diseases, making the aberrant glycan structures possible biomarkers [16,17]. Furthermore, in some diseases, the glycome differences are observed as changes in the abundance of specific glycans.…”

“…The results showed a high abundance of sialylated glycan isomers along with significant expression alterations in the brain cancer cell line and the breast cancer cell lines capable of brain metastasis, suggesting a relationship between sialylated glycan isomers and breast cancer brain metastasis. Moreover, they found that an increase of α2,6 linkage favors the passage of breast cancer cells through the blood–brain barrier, facilitating this type of cancer [16]. Anugraham et al.…”