Dataset Information

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

ABSTRACT: Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture and isobaric Tandem Mass Tag labeling enabled up to 32 single cells to be analyzed in a single LC-MS analysis, in addition to reference and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ~280 single cells were analyzed per day, which could be increased to ~700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions. Despite some decrease in coverage, this method readily differentiated four different cell types and thus proves suitable for, e.g., rapid cell typing.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Lymphoblast, Fibroblast

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD040455 | Pride | 2023-07-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	10ng_A549_H_Iso16_30CountFromKit.raw	Raw
	10ng_A549_L_Iso16_30CountFromKit.raw	Raw
	10ng_HeLa_H_Iso16_30CountFromKit.raw	Raw
	10ng_HeLa_L_Iso16_30CountFromKit.raw	Raw
	Chip1_K562HeLa_F2.raw	Raw

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HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics.

Liang Yiran Y Truong Thy T Saxton Aubrianna J AJ Boekweg Hannah H Payne Samuel H SH Van Ry Pam M PM Kelly Ryan T RT

Analytical chemistry 20230511 20

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric ta ...[more]

PMID: 37167627

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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.