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Nanodroplet processing platform for deep, quantitative proteome profiling of 10-100 mammalian cells

ABSTRACT: Proteomic data from 10-100 cultured cells and single human islet cross sections (Healthy control)

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pancreatic Islet

SUBMITTER: Matthew Monroe

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD006847 | Pride | 2018-01-24

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	CulturedCells_parameters.txt	Txt
	CulturedCells_peptides.txt	Txt
	CulturedCells_proteinGroups.txt	Txt
	CulturedCells_summary.txt	Txt
	Islet_t1d_ct_parameters.txt	Txt

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Publications

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

Zhu Ying Y Piehowski Paul D PD Zhao Rui R Chen Jing J Shen Yufeng Y Moore Ronald J RJ Shukla Anil K AK Petyuk Vladislav A VA Campbell-Thompson Martha M Mathews Clayton E CE Smith Richard D RD Qian Wei-Jun WJ Kelly Ryan T RT

Nature communications 20180228 1

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <20 ...[more]

PMID: 29491378

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Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

Project description:Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200?nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

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An Aspergillus niger colony locally adapts its molecular responses to spatially separated substrates

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DeNovo Peptide Identification Deep Learning Training Set

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Nanowell-Mediated Two-Dimensional Liquid Chromatography Proteome Profiling of Mammalian Cells

Project description:Proteomic data from technical developments of a nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separation for in-depth proteome profiling of low-nanogram samples.

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Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells.

Project description:Extending proteomics to smaller samples can enable the mapping of protein expression across tissues with high spatial resolution and can reveal sub-group heterogeneity. However, despite the continually improving sensitivity of LC-MS instrumentation, in-depth profiling of samples containing low-nanogram amounts of protein has remained challenging due to analyte losses incurred during preparation and analysis. To address this, we recently developed nanodroplet processing in one pot for trace samples (nanoPOTS), a robotic/microfluidic platform that generates ready-to-analyze peptides from cellular material in ~200 nL droplets with greatly reduced sample losses. In combination with ultrasensitive LC-MS, nanoPOTS has enabled >3000 proteins to be confidently identified from as few as 10 cultured human cells and ~700 proteins from single cells. However, the nanoPOTS platform requires a highly skilled operator and a costly in-house-built robotic nanopipetting instrument. In this work, we sought to evaluate the extent to which the benefits of nanodroplet processing could be preserved when upscaling reagent dispensing volumes by a factor of 10 to those addressable by commercial micropipette. We characterized the resulting platform, termed microdroplet processing in one pot for trace samples (μPOTS), for the analysis of as few as ~25 cultured HeLa cells (4 ng total protein) or 50 μm square mouse liver tissue thin sections and found that ~1800 and ~1200 unique proteins were respectively identified with high reproducibility. The reduced equipment requirements should facilitate broad dissemination of nanoproteomics workflows by obviating the need for a capital-intensive custom liquid handling system.

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2017-07-11 | PXD006084 | Pride

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Project description:Proteomic data from technical developments of a nanoPOTS-based spatially resolved proteome profiling of <200 cells from tomato fruit pericarp.

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