Aberrant glycosylation has been linked to many different cancer types. In breast cancer metastasis to the brain the blood brain barrier, a region of the brain that regulates the entrance of ions, diseases, toxins, etc., fails to block breast cancer cells from crossing. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 212 statistically significant glycopeptides in 231BR compared to the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.

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.

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

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Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes.