Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition DOI Creative Commons
Valdemaras Petrosius, Pedro Aragon-Fernandez, Nil Üresin

и другие.

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Сен. 22, 2023

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount material encapsulated in a single cell however, raises significant technical challenges molecular profiling. Due extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as powerful tool facilitate proteome profiling from ultra-low amounts input, although further development needed realize its full potential. To this end, we carry out comprehensive orbitrap-based data-independent acquisition (DIA) for proteomics. Notably, find difference between optimal DIA methods high- low-load samples. We improve our low-input method relying on high-resolution MS1 quantification, thus enhancing sensitivity more efficiently utilizing available mass analyzer time. With input tailored method, are able accommodate long injection times high resolution, while keeping the scan cycle time low enough ensure robust quantification. Finally, demonstrate capability approach mouse embryonic stem culture conditions, showcasing global proteomes highlighting differences key metabolic enzyme expression subclusters.

Язык: Английский

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip DOI Creative Commons
Jongmin Jacob Woo, Sarah Williams, Lye Meng Markillie

и другие.

Nature Communications, Год журнала: 2021, Номер 12(1)

Опубликована: Окт. 29, 2021

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve recovery, operation robustness, processing throughput for isobaric-labeling-based scProteomics workflow. The N2 reduces reaction volume <30 nL increases capacity >240 microchip. tandem mass tag (TMT) pooling step simplified by adding microliter droplet the nanowells combine labeled samples. In analysis ~100 individual from three different cell lines, demonstrate that chip-based platform can robustly quantify ~1500 proteins reveal membrane markers. Our analyses also low abundance variations, suggesting proteome profiles are highly stable cultured under identical conditions.

Язык: Английский

Процитировано

136
Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments DOI Open Access
Laurent Gatto, Ruedi Aebersold, Jüergen Cox

и другие.

Nature Methods, Год журнала: 2023, Номер 20(3), С. 375 - 386

Опубликована: Март 1, 2023

Язык: Английский

Процитировано

104
Exploring functional protein covariation across single cells using nPOP DOI Creative Commons
Andrew Leduc, R. Gray Huffman, Joshua Cantlon

и другие.

Genome biology, Год журнала: 2022, Номер 23(1)

Опубликована: Дек. 16, 2022

Many biological processes, such as cell division cycle and drug resistance, are reflected in protein covariation across single cells. This can be quantified interpreted by single-cell mass spectrometry with sufficiently high throughput accuracy.

Язык: Английский

Процитировано

103
Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics DOI Creative Commons
Manuel Matzinger, Elisabeth Müller, Gerhard Dürnberger

и другие.

Analytical Chemistry, Год журнала: 2023, Номер 95(9), С. 4435 - 4445

Опубликована: Фев. 20, 2023

The analysis of ultralow input samples or even individual cells is essential to answering a multitude biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here, we report comprehensive workflow that includes improved strategies for all steps, from cell lysis data analysis. Thanks convenient-to-handle 1 μL sample volume standardized 384-well plates, the easy novice users implement. At same time, it can be performed semi-automatized using CellenONE, which allows highest To achieve high throughput, ultrashort gradient lengths down 5 min were tested advanced μ-pillar columns. Data-dependent acquisition (DDA), wide-window (WWA), data-independent (DIA), commonly used algorithms benchmarked. Using DDA, 1790 proteins covering dynamic range four orders magnitude identified single cell. DIA, proteome coverage increased more than 2200 single-cell level 20 active gradient. enabled differentiation two lines, demonstrating its suitability cellular heterogeneity determination.

Язык: Английский

Процитировано

87
Understanding tumour endothelial cell heterogeneity and function from single-cell omics DOI
Qun Zeng, Mira Mousa,

Aisha Shigna Nadukkandy

и другие.

Nature reviews. Cancer, Год журнала: 2023, Номер 23(8), С. 544 - 564

Опубликована: Июнь 22, 2023

Язык: Английский

Процитировано

86
Unidirectional single-file transport of full-length proteins through a nanopore DOI
Luning Yu, Xinqi Kang, Fanjun Li

и другие.

Nature Biotechnology, Год журнала: 2023, Номер 41(8), С. 1130 - 1139

Опубликована: Янв. 9, 2023

Язык: Английский

Процитировано

82
Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering DOI Creative Commons
Jongmin Jacob Woo, Gérémy Clair, Sarah Williams

и другие.

Cell Systems, Год журнала: 2022, Номер 13(5), С. 426 - 434.e4

Опубликована: Март 16, 2022

Single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge current methods is their inability identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition peptide identification method, transferring based on FAIMS filtering (TIFF), improve the sensitivity accuracy label-free scProteomics. TIFF extends ion accumulation times ions by out singly charged ions. The identities are assigned three-dimensional MS1 feature matching approach (retention time, mass, compensation voltage). method enabled unbiased proteome analysis depth >1,700 proteins in single HeLa cells, with >1,100 consistently identified. As demonstration, applied obtain temporal profiles >150 murine macrophage cells during lipopolysaccharide stimulation identified time-dependent changes. A record this paper's transparent peer review process included supplemental information.

Язык: Английский

Процитировано

72
Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics DOI Creative Commons
Benjamin Furtwängler, Nil Üresin,

Khatereh Motamedchaboki

и другие.

Molecular & Cellular Proteomics, Год журнала: 2022, Номер 21(4), С. 100219 - 100219

Опубликована: Фев. 25, 2022

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on Orbitrap Eclipse Tribrid mass spectrometer combination with SPS-MS3 acquisition has been shown to be beneficial measurement samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times high-resolution spectra quantify signal; however, carrier channel facilitates peptide identification thus offers opportunity fast on-the-fly precursor filtering before committing time-intensive quantification scan. Here, we compared classical MS2 against RTS-SPS-MS3, MS FAIMS Pro mobility interface present new strategy termed RETICLE (RTS enhanced quant single spectra) makes use searched linear trap scans preselect MS1 precursors acquisition. We show outperformed by RTS-SPS-MS3 through increased accuracy at similar coverage, higher latter enabling over 1000 an time 750 ms 2 h gradient.

Язык: Английский

Процитировано

72
Into the multiverse: advances in single-cell multiomic profiling DOI Creative Commons

Silvia Ogbeide,

Francesca Giannese, Laura Mincarelli

и другие.

Trends in Genetics, Год журнала: 2022, Номер 38(8), С. 831 - 843

Опубликована: Май 8, 2022

Single-cell transcriptomic approaches have revolutionised the study of complex biological systems, with routine measurement gene expression in thousands cells enabling construction whole-organism cell atlases. However, transcriptome is just one layer amongst many that coordinate to define type and state and, ultimately, function. In parallel widespread uptake single-cell RNA-seq (scRNA-seq), there has been a rapid emergence methods enable multiomic profiling individual cells, intercellular heterogeneity genome, epigenome, transcriptome, proteomes. Linking measurements from each these layers potential reveal regulatory functional mechanisms underlying behaviour healthy development disease.

Язык: Английский

Процитировано

71
Recent advances in proteomics and metabolomics in plants DOI Creative Commons
Shijuan Yan, Ruchika Bhawal, Zhibin Yin

и другие.

Molecular Horticulture, Год журнала: 2022, Номер 2(1)

Опубликована: Июль 23, 2022

Over the past decade, systems biology and plant-omics have increasingly become main stream in plant research. New developments mass spectrometry bioinformatics tools, methodological schema to integrate multi-omics data leveraged recent advances proteomics metabolomics. These progresses are driving a rapid evolution field of research, greatly facilitating our understanding mechanistic aspects metabolisms interactions plants with their external environment. Here, we review MS-based metabolomics tools workflows special focus on applications research using several case studies related stress response, gene/protein function characterization, metabolic signaling pathways exploration, natural product discovery. We also present projection concerning future perspectives development including challenges for system biology. This is intended provide readers an overview how advanced MS technology, integrated application can be used advance

Язык: Английский

Процитировано

70