Dataset Information

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

ABSTRACT: Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from na??ve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

SUBMITTER: Moehring F

PROVIDER: S-EPMC6195672 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Moehring Francie F Waas Matthew M Keppel Theodore R TR Rathore Deepali D Cowie Ashley M AM Stucky Cheryl L CL Gundry Rebekah L RL

Journal of proteome research 20180706 8

Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, protein ...[more]

PMID: 29925238

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Project description:Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.

Dataset Information

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Publications

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

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