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Analysis of nucleotide variants is a cornerstone of cancer medicine. Although only 2% of the genomic sequence is protein coding, mutations occurring in these regions have the potential to influence protein structure or modification status and may have severe impact on disease aetiology. Proteogenomics enables the analysis of sample-specific nonsynonymous nucleotide variants with regard to their effect at the proteome and phosphoproteome levels. Here, we developed a proof-of-concept proteogenomics workflow and applied it to the malignant melanoma cell line A375. Initially, we studied the resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in A375 cells. This allowed identification of several oncogenic nonsynonymous nucleotide variants, including a gain-of-function variant on aurora kinase A (AURKA) at F31I. We also detected significant changes in abundance among (phospho)proteins, which led to reactivation of the MAPK signaling pathway in BRAFi-resistant A375 cells. Upon reconstruction of the multiomic integrated signaling networks, we predicted drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that AURKA inhibition is effective and specific against BRAFi-resistant A375 cells. Subsequently, we investigated amino acid variants that interfere with protein posttranslational modification (PTM) status and potentially influence A375 cell signaling irrespective of BRAFi resistance. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins. Among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome., Graphical Abstract, Highlights • Proteogenomics workflow to study the malignant melanoma cell line A375. • Key signaling pathways are perturbed in BRAFi-sensitive and -resistant A375. • The signaling network in BRAFi-resistant cells can be targeted by several drugs. • Multiple amino acid variants directly affect protein phosphorylation status. • Loss of a phosphorylation site on RUNX1 alters its interactome., In Brief Proteogenomics is a powerful tool to study the mode of action of disease-associated mutations at the genome, transcriptome, proteome, and PTM level. Here, we applied a proteogenomics workflow to study the malignant melanoma cell line A375. Such workflow, used here as a proof of concept on A375 cells, may be applicable to other cancer types, cell lines, or even patient-derived samples.