Dataset Information

Rapid, One-step Sample Processing for Label-Free Single-Cell Proteomics

ABSTRACT: Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long workflows can be labor intensive and lead to long sample-to-answer times. Here we report a sample processing method that achieves cell lysis, protein denaturation and digestion in one hour with a single reagent dispensing step using commercially available high-temperature-stabilized proteases. Four different one-step reagent compositions were evaluated, and the mixture providing greatest proteome coverage was compared to the previously employed multi-step workflow. The one-step preparation increases proteome coverage relative to the multi-step workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene substrates, and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled identification of an average of nearly 2,400 proteins per cell using a standard data-dependent workflow with an Orbitrap mass spectrometer. As such, these advances significantly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Ryan Kelly

LAB HEAD: Dr Ryan T. Kelly

PROVIDER: PXD041879 | Pride | 2023-10-09

REPOSITORIES: Pride

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Publications

Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics.

Johnston S Madisyn SM Webber Kei G I KGI Xie Xiaofeng X Truong Thy T Nydegger Alissia A Lin Hsien-Jung L HL Nwosu Andikan A Zhu Ying Y Kelly Ryan T RT

Journal of the American Society for Mass Spectrometry 20230706 8

Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive and lead to long sample-to-answer times. Here we report a sample preparation method that achieves cell lysis, protein denaturation, and digestion in 1 h using commercially available high-temperature-stabilized proteases with a single reagent dispensing step. Four different one-step reagent compositions were eva ...[more]

PMID: 37410391

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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

Rapid, One-step Sample Processing for Label-Free Single-Cell Proteomics

Publications

Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics.

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