Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

• MeSH
• DNA MeSH
• Ions MeSH
• Isotopes MeSH
• Proteins * analysis   MeSH
• Tandem Mass Spectrometry * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Ions MeSH
• Isotopes MeSH
• Proteins * MeSH

Extracellular signal-regulated kinase (ERK) is a part of the mitogen-activated protein kinase (MAPK) signaling pathway which allows the transduction of various cellular signals to final effectors and regulation of elementary cellular processes. Deregulation of the MAPK signaling occurs under many pathological conditions including neurodegenerative disorders, metabolic syndromes and cancers. Targeted inhibition of individual kinases of the MAPK signaling pathway using synthetic compounds represents a promising way to effective anti-cancer therapy. Cross-talk of the MAPK signaling pathway with other proteins and signaling pathways have a crucial impact on clinical outcomes of targeted therapies and plays important role during development of drug resistance in cancers. We discuss cross-talk of the MAPK/ERK signaling pathway with other signaling pathways, in particular interplay with the Hippo/MST pathway. We demonstrate the mechanism of cell death induction shared between MAPK/ERK and Hippo/MST signaling pathways and discuss the potential of combination targeting of these pathways in the development of more effective anti-cancer therapies.

• Keywords
• ERK, Hippo, MAPK, MST, PI3K, YAP, apoptosis, cancer, caspase, inhibitors, natural compounds, therapy,
• MeSH
• Extracellular Signal-Regulated MAP Kinases antagonists & inhibitors   metabolism   MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Humans MeSH
• Neoplasms drug therapy   metabolism   MeSH
• Protein Serine-Threonine Kinases antagonists & inhibitors   metabolism   MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Hippo Signaling Pathway MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Extracellular Signal-Regulated MAP Kinases MeSH
• Protein Kinase Inhibitors MeSH
• Protein Serine-Threonine Kinases MeSH
• Antineoplastic Agents MeSH