Comparative Surface Proteomic Analysis Of Human Breast Cancer Cell Lines

Enrichment of cell surface proteins is commonly performed by biotinylation using amine-based chemistry; however, this method is often associated with intracellular labeling and difficulty distinguishing the true cell surface proteins from background contaminants. We developed a robust cell surface protein isolation method using amine-based chemistry that is compatible with mass spectrometry (MS). We applied this methodology to evaluate the surface proteomes of three breast cancer cell lines, including hormone responsive MCF-7 cells, and two triple negative breast cancer cell lines (TNBC), BT549 and Hs5789T. Thermo Scientific™ Tandem Mass Tag (TMT) Reagents, combined with high pH reverse phase fractionation of the labeled peptides, were used to multiplex protein identification and quantitative analyses by tandem MS. A heat map of enriched proteins from the three cell lines showed clearly distinct banding patterns of clustered protein groups. Relative quantitative differences in several classes of enriched proteins were observed, including tyrosine kinases, G-Protein-Coupled Receptors (GPCRs), and cell adhesion proteins. The tyrosine kinase EGFR was found to be more highly expressed in TNBC cell lines compared with MCF-7 cells. In contrast, the receptor tyrosine-protein kinase erbB-3 was found to be significantly higher in the MCF-7 cells compared with the TNBC cells. Several GPCRs were identified and found to vary across the three cell lines, including Frizzled 7 which is an emerging target for cancer therapy. Multiple cell adhesion proteins were also identified, including neural cell adhesion molecule L1 which promotes migration of MCF7 cells. Where possible, the data set was found to be consistent with literature reports. In conclusion, we have developed a highly sensitive method for cell surface proteomic analyses that can be effectively used to identify differences in cell types as well as for biomarker discovery and potential drug therapy targets. Citation Format: Betsy Benton, Sergei Snovida, Katherine Herting, Kay Opperman, Barbara Kaboord. Comparative surface proteomic analysis of human breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2858.