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Electrophoresis-Correlative Framing of Ion Mobility Deepens Single-Cell Mass Spectrometry Proteomics

ABSTRACT: This project developed electrophoresis-correlative (Eco) mass spectrometry (MS) to measure the proteome of single cells on a trapped ion mobility mass spectrometer (timsTOF PRO). Eco-MS identified 962 protein groups in a <20 min of electrophoresing a single-HeLa-cell-equivalent (~200 pg) proteome amount. Match-between-runs improved this number to 2,139 protein groups on the same dataset. We demonstrate the approach to measure the proteome of single cells in X. laevis embryos at stage 8 (blastula). Single cells were isolated from the animal cap and their proteomes digested on a fluorosilane-coated microplate. Less than 4% of the available single-cell proteome was analyzed on a custom-built capillary electrophoresis (CE) platform under classical and Eco-driven ddaPASEF. Eco-MS returned ~51% of more protein groups than the control, identifying 1,157 proteins from N = 9 different single cells (biological replicates) of the animal cap. Eco-MS deepens the detectable coverage of the single-cell proteome using ddaPASEF on a timsTOF MS.

INSTRUMENT(S): timsTOF

ORGANISM(S): Homo Sapiens (human) Xenopus Laevis (african Clawed Frog)

TISSUE(S): Cell Culture

SUBMITTER: Peter Nemes

LAB HEAD: Peter Nemes

PROVIDER: PXD053023 | Pride | 2025-02-06

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	200pgHeLa_processing.zip	Other
	200pgHeLa_raw.zip	Other
	2023-05-13-decoys-contam-2023-Human.fasta.fas	Fasta
	500pgHeLa_dataProcessing.zip	Other
	500pgHeLa_raw.zip	Other

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Project description:Purpose: We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Experimental design: Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias. Results: In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate â¤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue. Conclusions and clinical relevance: These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics. Affymetrix GeneChip Mouse Genome 430 2.0 microarrays were used to profile whole tissues from 5 different tissue types of 25 tumor-bearing and 25 control mice of the Her2/Neu breast cancer mouse model. The 5 tissues tested were from breast, liver, spleen, blood cell, and thymus. The tumor-bearing mice were bitransgenic for MMTV-rtTA/TetO-NeuNT, and the control mice were transgenic for TetO-NeuNT only. The control mice were age- and cage-matched to the tumor-bearing mice. All samples were lysed and total RNA isolated and amplified prior to hybridization.

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Electrophoresis-Correlative Framing of Ion Mobility Deepens Single-Cell Mass Spectrometry Proteomics

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