Dataset Information

High-resolution spatially-resolved proteome mapping using automated, sacrificial liquid-mediated sample transfer from laser microdissection to nanodroplet sample preparation

ABSTRACT: Method development for high-resolution spatially-resolved proteome mapping of tissue heterogeneity. Laser microdissection was coupled with nanodroplet sample preparation and ultrasensitive LC-MS.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Matthew Monroe

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD008844 | Pride | 2018-07-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MaxQuant_Parameters.txt	Txt
	MaxQuant_Peptides.txt	Txt
	MaxQuant_ProteinGroups.txt	Txt
	MaxQuant_Summary.txt	Txt
	Ying_DMSO_RatBrain_100um_1_082217Prep.mgf	Mgf

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Publications

Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets.

Zhu Ying Y Dou Maowei M Piehowski Paul D PD Liang Yiran Y Wang Fangjun F Chu Rosalie K RK Chrisler William B WB Smith Jordan N JN Schwarz Kaitlynn C KC Shen Yufeng Y Shukla Anil K AK Moore Ronald J RJ Smith Richard D RD Qian Wei-Jun WJ Kelly Ryan T RT

Molecular & cellular proteomics : MCP 20180624 9

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Tra ...[more]

PMID: 29941660

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Project description:Background:Haematoxylin and eosin (H&E)-which respectively stain nuclei blue and other cellular and stromal material pink-are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Methods:Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n?=?3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. Results:This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3. Conclusion:Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues.