Dataset Information

Diversity of post-translational modifications and signaling networks revealed by single cell and organelle mass spectrometry

ABSTRACT: Using an optimized data-independent acquisition approach on trapped ion mobility spectrometry, we measured proteins, post-translational modifications and variant peptides in single cells and single nuclei, which provided insights into heterogeneity of cell-state and signaling dependencies at the single cell level and the molecular impact of an epigenetic drug at the level of a single organelle.

INSTRUMENT(S): timsTOF SCP

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cholangiocyte, T Cell

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD044986 | Pride | 2024-08-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CCACells_DDA.zip	Other
	CCA_SCP_DIA_Result.zip	Other
	EGI_20C_DDA_R1_IO_L15_Slot2-37_1_5042.d.tar.gz	Other
	EGI_20C_DDA_R2_IO_L15_Slot2-38_1_5043.d.tar.gz	Other
	EGI_20C_DDA_R3_IO_L15_Slot2-39_1_5044.d.tar.gz	Other

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Publications

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

Mun Dong-Gi DG Bhat Firdous A FA Joshi Neha N Sandoval Leticia L Ding Husheng H Jain Anu A Peterson Jane A JA Kang Taewook T Pujari Ganesh P GP Tomlinson Jennifer L JL Budhraja Rohit R Zenka Roman M RM Kannan Nagarajan N Kipp Benjamin R BR Dasari Surendra S Gaspar-Maia Alexandre A Smoot Rory L RL Kandasamy Richard K RK Pandey Akhilesh A

Communications biology 20240719 1

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuc ...[more]

PMID: 39030393

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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.