Dataset Information

Automated coupling of nanodroplet sample preparation with liquid chromatography-mass spectrometry for high throughput single-cell proteomics

ABSTRACT: Development and evaluation of an autosampler for integrating nanoPOTS with LC-MS. Includes proteomic data from nanoPOTS-based sample preparation, including diluted peptides of Shewanella oneidensis MR-1, single cultured MCF10A cells, and single cells from three Acute Myeloid Leukemia (AML) cell lines (MOLM-14, K562, and CMK). Data was searched with MaxQuant.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Shewanella Oneidensis Mr-1 (ncbitaxon:211586) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ying Zhu

PROVIDER: MSV000085230 | MassIVE | Sun Apr 05 09:44:00 BST 2020

REPOSITORIES: MassIVE

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Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics.

Williams Sarah M SM Liyu Andrey V AV Tsai Chia-Feng CF Moore Ronald J RJ Orton Daniel J DJ Chrisler William B WB Gaffrey Matthew J MJ Liu Tao T Smith Richard D RD Kelly Ryan T RT Pasa-Tolic Ljiljana L Zhu Ying Y

Analytical chemistry 20200722 15

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challe ...[more]

PMID: 32639140

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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.