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Isobaric peptide labeling on digital microfluidics for quantitative proteomics of low cell numbers is facilitated by low critical micellar concentration detergents

ABSTRACT: We developed protocols for isobaric TMT labeling on digital microfluidics (DMF) devices enabling the quantitative proteome analysis of approximately 25 mammalian cells per channel by subsequent bottom-up proteome analysis by LC-MS. This comprised identification of a compatible detergent for digestion, on-chip labeling, and LC-MS. Application of the method in a TMT 6-plex sample analysis enabled the relative quantification 648 proteins (2 unique peptides for quantification) from a total of 125 cell equivalents.

INSTRUMENT(S): Q Exactive Plus, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Andreas Tholey

LAB HEAD: Prof. Dr. Andreas Tholey

PROVIDER: PXD023226 | Pride | 2022-02-28

REPOSITORIES: pride

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			Action	DRS
	110820_1kjurkats_SP3tube_BBS-mix_6hrs_1u_100cells_R1.pdResult	Other
	110820_1kjurkats_SP3tube_BBS-mix_6hrs_1u_100cells_R1.raw	Raw
	110820_1kjurkats_SP3tube_BBS-mix_6hrs_1u_100cells_R2.pdResult	Other
	110820_1kjurkats_SP3tube_BBS-mix_6hrs_1u_100cells_R2.raw	Raw
	110820_1kjurkats_SP3tube_BBS-mix_6hrs_1u_100cells_R3.pdResult	Other

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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.

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Isobaric peptide labeling on digital microfluidics for quantitative proteomics of low cell numbers is facilitated by low critical micellar concentration detergents

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