Dataset Information

Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

ABSTRACT: Mass spectrometry imaging is a powerful tool of increasing utility due to its ability to spatially resolve molecular biomarkers directly from sectioned tissues. One hindrance to its universality is that no single protocol is sufficient for every tissue type, fixation, and pretreatment. Mineralized tissues are uniquely challenging as extensive decalcification protocols are necessary to achieve thin sections. In this study, we optimized a method to image tryptic peptides by matrix-assisted laser desorption ionization mass spectrometry of decalcified, formalin-fixed paraffin-embedded mouse hemimandibles. Using a combination of on-tissue MS/MS and hydrogel extraction LC-MS/MS, peptides from the enamel, dentin, periodontal ligament, alveolar bone, pulp, and other regions are identified and mapped. This breakthrough method provides a comprehensive approach to biomarker discovery in dental and craniofacial tissues which is highly relevant given that diseases originating from this region of the body are the most prevalent across all populations.

SUBMITTER: Colley M

PROVIDER: S-EPMC7898936 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

Colley Madeline M Liang Sitai S Tan Chunyan C Trobough Kyle P KP Bach Stephan B H SBH Chun Yong-Hee Patricia YP

Analytical chemistry 20200512 11

Mass spectrometry imaging is a powerful tool of increasing utility due to its ability to spatially resolve molecular biomarkers directly from sectioned tissues. One hindrance to its universality is that no single protocol is sufficient for every tissue type, fixation, and pretreatment. Mineralized tissues are uniquely challenging as extensive decalcification protocols are necessary to achieve thin sections. In this study, we optimized a method to image tryptic peptides by matrix-assisted laser d ...[more]

PMID: 32362116

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Project description:BackgroundThyroid hormones play an essential role in early vertebrate development as well as other key processes. One of its modes of action is to bind to the thyroid hormone receptor (TR) which, in turn, binds to thyroid response elements (TREs) in promoter regions of target genes. The sequence motif for TREs remains largely undefined as does the precise chromosomal location of the TR binding sites. A chromatin immunoprecipitation on microarray (ChIP-chip) experiment was conducted using mouse cerebellum post natal day (PND) 4 and PND15 for the thyroid hormone receptor (TR) beta 1 to map its binding sites on over 5000 gene promoter regions. We have performed a detailed computational analysis of these data.ResultsBy analysing a recent spike-in study, the optimal normalization and peak identification approaches were determined for our dataset. Application of these techniques led to the identification of 211 ChIP-chip peaks enriched for TR binding in cerebellum samples. ChIP-PCR validation of 25 peaks led to the identification of 16 true positive TREs. Following a detailed literature review to identify all known mouse TREs, a position weight matrix (PWM) was created representing the classic TRE sequence motif. Various classes of promoter regions were investigated for the presence of this PWM, including permuted sequences, randomly selected promoter sequences, and genes known to be regulated by TH. We found that while the occurrence of the TRE motif is strongly correlated with gene regulation by TH for some genes, other TH-regulated genes do not exhibit an increased density of TRE half-site motifs. Furthermore, we demonstrate that an increase in the rate of occurrence of the half-site motifs does not always indicate the specific location of the TRE within the promoter region. To account for the fact that TR often operates as a dimer, we introduce a novel dual-threshold PWM scanning approach for identifying TREs with a true positive rate of 0.73 and a false positive rate of 0.2. Application of this approach to ChIP-chip peak regions revealed the presence of 85 putative TREs suitable for further in vitro validation.ConclusionsThis study further elucidates TRβ gene regulation in mouse cerebellum, with 211 promoter regions identified to bind to TR. While we have identified 85 putative TREs within these regions, future work will study other mechanisms of action that may mediate the remaining observed TR-binding activity.

Dataset Information

Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

Publications

Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

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