In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors

Abstract

Native intact N-glycopeptide analysis can provide access to the comprehensive characteristics of N-glycan occupancy, including N-glycosites, N-glycan compositions, and N-glycoproteins for complex samples. The sample pre-processing method used for the analysis of intact N-glycopeptides usually depends on the enrichment of low abundance N-glycopeptides from a tryptic peptide mixture using hydrophilic substances before LC-MS/MS detection. However, the number of identified intact N-glycopeptides remains inadequate to achieve an in-depth profile of the N-glycosylation landscape. Here, we optimized the sample preparation workflow prior to LC-MS/MS analysis by systematically comparing different analytical methods, including the use of different sources of trypsin, combinations of different proteases, and different enrichment materials. Finally, we found that the combination of Trypsin (B)/Lys-C digestion and zwitterionic HILIC (Zic-HILIC) enrichment significantly improved the mass spectrometric characterization of intact N-glycopeptides, increasing the number of identified intact N-glycopeptides and offering better analytical reproducibility. Furthermore, the optimized workflow was applied to the analysis of intact N-glycopeptides in two-dimensional (2D) and three-dimensional (3D)-cultured breast cancer cells in vitro and xenografted tumors in mice. These results indicated that the same breast cancer cells, when cultured in different microenvironments, can show different N-glycosylation patterns. This study also provides an interesting comparison of the N-glycoproteome of breast cancer cells cultured in different growth conditions, indicating the important role of N-glycosylated proteins in cancer cell growth and the choice of the cell culture model for studies in tumor biology and drug evaluation.

Cracking the Breast Cancer Glyco-Code through Glycan-Lectin Interactions: Targeting Immunosuppressive Macrophages

“…David and coworkers reported that 3D cultures of ovarian cancer cell lines showed a higher resemblance in terms of mucin and truncated O-glycan footprint to tumour tissue than the same cell lines cultured as 2D cultures [ 96 ]. Other reports followed in gastric and breast cancer [ 97 , 98 ]. For the latter, the authors describe different N-glycosylation patterns for the same BC cells, when cultured in 2D, 3D and as mouse xenografts [ 98 ].…”

“…Other reports followed in gastric and breast cancer [ 97 , 98 ]. For the latter, the authors describe different N-glycosylation patterns for the same BC cells, when cultured in 2D, 3D and as mouse xenografts [ 98 ].…”

Understanding the Role of Fibroblasts following a 3D Tumoroid Implantation for Breast Tumor Formation

“…Studies have shown that cells involved in a tumoroid perform metabolic and physical activities similar to what they do in native tissue. The ECM proteins produced by cells during tumoroid formation bind each other and form the complex networks that represent the tumor complexity as seen in xenografted tumors [ 117 , 118 ]. The methodologies to generate tumoroids are beyond the scope of this paper.…”

Construction of boric acid‐functionalized metal–organic frameworks for glycopeptide recognition in the serum of cervical cancer patients

“…Glycosylation, a template‐free enzymatic process, is one of the most common protein post‐translational modifications in human cell lines 1 . At the same time, glycopeptides carry a wealth of personal biological information, and play a key role in various fundamental biological processes such as immune regulation, cell growth, cell–cell interaction and cell adhesion 2–4 .…”

“…Glycosylation, a template-free enzymatic process, is one of the most common protein post-translational modifications in human cell lines. 1 At the same time, glycopeptides carry a wealth of personal biological information, and play a key role in various fundamental biological processes such as immune regulation, cell growth, cell-cell interaction and cell adhesion. [2][3][4] Abnormal glycosylation is closely related to the progress of many tumor-related diseases; for instance, glycosylation disorders exist in the development and progression of cervical cancer.…”