Spatially resolved characterization of proteoforms for functional proteomics
功能蛋白质组学蛋白质型的空间分辨表征
基本信息
- 批准号:10118771
- 负责人:
- 金额:$ 30万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-09-08 至 2022-08-31
- 项目状态:已结题
- 来源:
- 关键词:3-DimensionalAcheAddressAmino Acid SequenceAreaAwarenessBioinformaticsBiologyBladderCell Differentiation processCell SeparationCell physiologyCellsChromatinChromatographyComplexCouplingCustomDNA MethylationDataDatabasesDetectionDevelopmentDiabetic NephropathyDiseaseDissociationEnd stage renal failureEndothelial CellsEpigenetic ProcessExposure toFourier TransformGenesGoalsHealthHistonesHumanHuman BioMolecular Atlas ProgramImageKidneyLasersLiquid substanceLiverLocationMapsMass Spectrum AnalysisMeasurementMeasuresMethodsMicrofluidicsMolecularMorphologyMultimodal ImagingNucleosomesOrganPathogenesisPatternPeptidesPhasePlayPost-Translational Protein ProcessingPreparationProcessProteinsProteolysisProteomeProteomicsRNA methylationRecoveryResearchResolutionRoleSamplingSeriesSideSourceSystemTechnologyTissue imagingTissuesToxic Environmental SubstancesTranscriptional RegulationUntranslated RNAVariantVisualization softwareWorkbasebioinformatics toolcombinatorialcommercializationcostdata integrationdata visualizationdesignhigh throughput analysishistone modificationhuman tissueimage processingimage visualizationimaging approachimaging modalityimprovedinnovationinterestlaser capture microdissectionmesangial cellmultimodal datamultimodalitynanoDropletnext generationnovelopen sourcepodocyteprogramsstoichiometrytooltranscriptomicsvirtual
项目摘要
PROJECT SUMMARY/ABSTARCT
Differentiated cells have distinctive patterns of epigenetic marks including various post-translational
modifications (PTMs) on histones that may work in concert to control transcriptional programs. Since epigenetic
marks are often altered following exposure to environmental toxins and play multiple roles in disease
pathogenesis, the ability to measure histones in a tissue and cell context is a major analytical objective and
challenge. Mass spectrometry (MS) based proteomics is a powerful tool for characterizing histone alterations in
multiplexed and non-targeted fashion. However, conventional bottom-up (i.e. peptide-level) MS cannot provide
complete characterization of the stoichiometry and combinations of multiple PTMs, and other combinatorial
sources of variation, that collectively make up any single gene's set of proteoforms (i.e. functional units of a
proteome). Top-down (i.e. proteoform-level) MS addresses this challenge by omitting the proteolysis and thus
allowing access to the functional proteoforms. However, top-down MS suffers from low sensitivity and dynamic
range due to challenges in separation and detection of large and low-abundance proteins and laborious
purification steps required to achive high proteome coverage. This severely limits our ability to analyze small
samples and employ top-down MS to generate proteoform-aware images of tissues required for a deeper
understanding of human organ functioning in health and disease. We have recently developed nanodroplet
sample preparation (nanoPOTS) for highly sensitive bottom-up proteomics and extended this approach to tissue
imaging with 100 µm spatial resolution. Herein, we propose to develop and deploy nanoPOTS-based top-down
MS to enable characterization of proteoforms in tissue sections with near single cell resolution. To increase the
resolution from thousands of cells to near single cell, we will employ advanced MS imaging (MSI) approaches.
MSI data will be cross-referenced with global proteomics data obtained via microscale top-down MS of
microdissected tissue regions. The UG3 phase efforts will be focused on histones and kidney as a development
platform and leverage a unique combination of microscale top-down LCMS, MSI and novel image processing
and visualization tools. In the UH3 phase, we will construct comprehensive proteoform-specific maps of multiple
tissue types and facilitate multimodal molecular mapping of specific functional units of the kidney by leveraging
the HubMAP Consortium ongoing efforts. Successful completion of this research will allow for comprehensive
characterization of the full spectrum of proteoforms in tissues and cells thus addressing an important and under-
studied area of biology and critical gap in HuBMAP efforts.
项目摘要/戒律
分化细胞具有独特的表观遗传标记模式,包括各种翻译后
对组蛋白的修改(PTM)可能会协同控制转录程序。自表观遗传学以来
暴露于环境毒素后通常会改变痕迹,并在疾病中发挥多重作用
发病机理,在组织和细胞环境中测量组蛋白的能力是一个主要的分析目的,
挑战。基于质谱(MS)的蛋白质组学是表征组蛋白改变的强大工具
多路复用和非目标的时尚。但是,传统的自下而上(即肽级)MS无法提供
多个PTM和其他组合的化学计量和组合的完整表征
变异的来源,共同构成了任何单个基因的蛋白质成型集合(即
蛋白质组)。自上而下(即蛋白质成型级)MS通过省略蛋白水解,从而解决这一挑战
允许访问功能蛋白基础。但是,自上而下的MS遭受低灵敏度和动态性
由于分离和检测大量和低丰度蛋白的挑战以及费力的范围
实现高蛋白质组覆盖范围所需的纯化步骤。这严重限制了我们分析小的能力
样本和员工自上而下的MS,以生成更深层次的时间的蛋白质成型图像
了解人体器官在健康和疾病中的功能。我们最近开发了纳米词
样品制备(纳米植物)用于高度敏感的自下而上蛋白质组学,并将这种方法扩展到组织
用100 µM空间分辨率进行成像。本文中,我们建议开发和部署基于纳米机器的自上而下
MS能够表征具有接近单细胞分辨率的组织切片中蛋白成型的表征。增加
从数千个细胞到近单元的分辨率,我们将采用高级MS成像(MSI)方法。
MSI数据将与通过微观自上而下的MS获得的全局蛋白质组学数据交叉引用
微解剖的组织区域。 UG3阶段努力将集中于组蛋白和肾脏作为发展
平台并利用微观自上而下的LCM,MSI和新型图像处理的独特组合
和可视化工具。在UH3阶段,我们将构建多个多重蛋白质成型图特异性地图
通过利用组织类型并促进肾脏特定功能单元的多模式分子图。
Hubmap联盟正在进行的努力。成功完成这项研究将允许全面
表征组织和细胞中蛋白质成型的全光谱,从而解决了重要和不足
在Hubmap工作中研究生物学领域和关键差距。
项目成果
期刊论文数量(0)
专著数量(0)
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会议论文数量(0)
专利数量(0)
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Ljiljana Pasa-Tolic的其他文献
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{{ truncateString('Ljiljana Pasa-Tolic', 18)}}的其他基金
Massive single cell proteomics for cancer biology
用于癌症生物学的大规模单细胞蛋白质组学
- 批准号:
10707321 - 财政年份:2022
- 资助金额:
$ 30万 - 项目类别:
Spatially-resolved proteome mapping of senescent cells and their tissue microenvironment at single-cell resolution
单细胞分辨率下衰老细胞及其组织微环境的空间分辨蛋白质组图谱
- 批准号:
10684865 - 财政年份:2022
- 资助金额:
$ 30万 - 项目类别:
Spatially-resolved proteome mapping of senescent cells and their tissue microenvironment at single-cell resolution
单细胞分辨率下衰老细胞及其组织微环境的空间分辨蛋白质组图谱
- 批准号:
10552842 - 财政年份:2022
- 资助金额:
$ 30万 - 项目类别:
Spatially resolved characterization of proteoforms for functional proteomics
功能蛋白质组学蛋白质型的空间分辨表征
- 批准号:
10687330 - 财政年份:2020
- 资助金额:
$ 30万 - 项目类别:
Spatially resolved characterization of proteoforms for functional proteomics
功能蛋白质组学蛋白质型的空间分辨表征
- 批准号:
10889043 - 财政年份:2020
- 资助金额:
$ 30万 - 项目类别:
Spatially resolved characterization of proteoforms for functional proteomics
功能蛋白质组学蛋白质型的空间分辨表征
- 批准号:
10256724 - 财政年份:2020
- 资助金额:
$ 30万 - 项目类别:
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