Dataset Information

A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC)-Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry

ABSTRACT: We developed a novel membrane-permeable and IMAC-enrichable cross-linker tBu-PhoX. Applying our optimized in vivo XL-MS pipeline, we revealed protein interaction landscape of intact human cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Heart, Cell Culture

SUBMITTER: Pin-Lian Jiang

LAB HEAD: Fan Liu

PROVIDER: PXD029034 | Pride | 2022-01-11

REPOSITORIES: Pride

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Publications

A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry.

Jiang Pin-Lian PL Wang Cong C Diehl Anne A Viner Rosa R Etienne Chris C Nandhikonda Premchendar P Foster Leigh L Bomgarden Ryan D RD Liu Fan F

Angewandte Chemie (International ed. in English) 20220127 12

Cross-linking mass spectrometry (XL-MS) is an attractive method for the proteome-wide characterization of protein structures and interactions. Currently, the depth of in vivo XL-MS studies is lagging behind the established applications to cell lysates, because cross-linking reagents that can penetrate intact cells and strategies to enrich cross-linked peptides lack efficiency. To tackle these limitations, we have developed a phosphonate-containing cross-linker, tBu-PhoX, that efficiently permeat ...[more]

PMID: 34927332

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Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

Dataset Information

A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC)-Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry

Publications

A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross-Linking Reagent to Advance In Vivo Cross-Linking Mass Spectrometry.

Similar Datasets

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