Dataset Information

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants

ABSTRACT: We used digital microfluidics (DMF) for the sample preparation of approximately 100 mammalian cells, and subsequent bottom-up proteome analysis by LC-MS. This comprised optimization of cell lysis conditions for DMF, the development of detergent-buffer systems, and adaptation of the single-pot, solid-phase-enhanced sample preparation (SP3) approach on-chip. Application of the methodology to the proteome analysis of Jurkat T cells led to the identification of up to 2,500 proteins from approximately 500 cells, and up to 1,200 proteins from approximately 100 cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, T Cell

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD014158 | Pride | 2019-10-07

REPOSITORIES: Pride

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Publications

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants.

Leipert Jan J Tholey Andreas A

Lab on a chip 20191001 20

While LC-MS-based proteomics with high nanograms to micrograms of total protein has become routine, the analysis of samples derived from low cell numbers is challenged by factors such as sample losses, or difficulties encountered with the manual manipulation of small liquid volumes. Digital microfluidics (DMF) is an emerging technique for miniaturized and automated droplet manipulation, which has been proposed as a promising tool for proteomic sample preparation. However, proteome analysis of sa ...[more]

PMID: 31531506

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Project description:The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

Dataset Information

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants

Publications

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants.

Similar Datasets

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