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Malignant melanoma is an aggressive type of skin cancer, rarely detected in the early stages. Various sets of methods and techniques, including dermatoscopical inspection of the "ABCDE" signs of the lesion, imaging techniques or microscopical, immunohistochemical and serological biomarkers are available and used nowadays to diagnose malignant melanoma. To date, different biomarkers were proposed for melanoma, but only a few, including circulating proteins, such as lactate dehydrogenase, molecular and metabolite biomarkers, have reached clinical applications. Gangliosides represent an emerging class, being used as tumor markers and targets of antibody therapy in melanomas, based on their elevated abundance in melanoma, especially of GM3 and GD3, when compared with the corresponding normal tissues. The conjunction of mass spectrometry (MS) with ion mobility separation (IMS) demonstrated an elevated potential in detection and identification of low abundant components, with biomarker role, in extremely complex biological mixtures. Therefore, here, a native ganglioside extract originating from human melanoma was investigated for the first time by IMS MS to provide the first profiling of gangliosides in this type of cancer.