An optimized isolation of biotinylated cell surface proteins reveals novel players in cancer metastasis

Details of metastasis, the deadliest aspect of cancer, are unclear. Cell surface proteins play central roles in adhesive contacts between the tumor cell and the stroma during metastasis. We optimized a fast, small-scale isolation of biotinylated cell surface proteins to reveal novel metastasis-associated players from an isogenic pair of human MDA-MB-435 cancer cells with opposite metastatic phenotypes. Isolated proteins were trypsin digested and analyzed using LC–MS/MS followed by quantitation with the Progenesis LC–MS software. Sixteen proteins displayed over twofold expression differences between the metastatic and non-metastatic cells. Interestingly, overexpression of most of them (14/16) in the metastatic cells indicates a gain of novel surface protein profile as compared to the non-metastatic ones. All five validated, differentially expressed proteins showed higher expression in the metastatic cells in culture, and four of these were further validated in vivo. Moreover, we analyzed expression of two of the identified proteins, CD109 and ITGA6 in 3-dimensional cultures of six melanoma cell lines. Both proteins marked the surface of cells derived from melanoma metastasis over cells derived from primary melanoma. The unbiased identification and validation of both known and novel metastasis-associated proteins indicate a reliable approach for the identification of differentially expressed surface proteins.

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► Optimized protocol for successful isolation of biotinylated cell surface proteins.
► Quantitative comparison of metastatic and non-metastatic cell surface proteins.
► Most proteins overexpressed in the metastatic cells indicating gain‐of-function.
► Pathway analyses.
► CD109 as a novel metastasis‐associated protein.