Single-cell proteomics enabled by next-generation sequencing or mass spectrometry

Nature Methods, Год журнала: 2023, Номер 20(3), С. 363 - 374

Опубликована: Март 1, 2023

Язык: Английский

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Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments

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

Опубликована: Март 1, 2023

Язык: Английский

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Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering

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

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Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

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

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An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity

Molecular & Cellular Proteomics, Год журнала: 2023, Номер 22(12), С. 100665 - 100665

Опубликована: Окт. 14, 2023

Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs instrumentation demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce proteoCHIP, a universal option for proteomics including multiplexed labeling up 16-plex high sensitivity throughput. The automated processing using commercial system combining isolation picoliter dispensing, cellenONE®, reduces final volumes low nanoliters submerged in hexadecane layer simultaneously eliminating error-prone manual handling overcoming evaporation. proteoCHIP design allows direct injection via standard autosampler resulting around 1,500 protein groups per TMT10-plex reduced or eliminated need carrier proteome. We evaluated effect wider precursor windows at input levels found that 2 Da increased overall without significantly impacting interference. Using dedicated MS acquisition strategies detailed here, identified on average close 2,000 proteins across 170 readily distinguished human types. Overall, our workflow combines highly preparation, chromatographic ion mobility-based filtering, rapid wide-window DDA analysis intelligent data optimal proteomics. This versatile proteoCHIP-based approach is sufficiently sensitive drive biological applications can be adopted by laboratories.

Язык: Английский

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Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition

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

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A review of the current state of single-cell proteomics and future perspective

Analytical and Bioanalytical Chemistry, Год журнала: 2023, Номер 415(28), С. 6889 - 6899

Опубликована: Июнь 7, 2023

Abstract Single-cell methodologies and technologies have started a revolution in biology which until recently has primarily been limited to deep sequencing imaging modalities. With the advent subsequent torrid development of single-cell proteomics over last 5 years, despite fact that proteins cannot be amplified like transcripts, it now become abundantly clear is worthy complement transcriptomics. In this review, we engage an assessment current state art including workflow, sample preparation techniques, instrumentation, biological applications. We investigate challenges associated with working very small volumes acute need for robust statistical methods data interpretation. delve into what believe promising future research at resolution highlight some exciting discoveries already made using proteomics, identification rare cell types, characterization cellular heterogeneity, investigation signaling pathways disease mechanisms. Finally, acknowledge there are number outstanding pressing problems scientific community vested advancing technology needs resolve. Of prime importance set standards so becomes widely accessible allowing novel easily verifiable. conclude plea solve these rapidly can part robust, high-throughput, scalable multi-omics platform ubiquitously applied elucidating insights diagnosis treatment all diseases afflict us.

Язык: Английский

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Pick-up single-cell proteomic analysis for quantifying up to 3000 proteins in a Mammalian cell

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Фев. 10, 2024

Abstract The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell still insufficient for practical applications. Here, we develop a pick-up (PiSPA) workflow to achieve deep capable quantifying up 3000 groups in mammalian using label-free quantitative method. PiSPA specially established samples mainly based on nanoliter-scale microfluidic liquid handling robot, achieving capture, pretreatment and injection under operation strategy. Using this customized with remarkable improvement identification, 2449–3500, 2278–3257 1621–2904 are quantified single A549 cells ( n = 37), HeLa 44) U2OS 27) DIA (MBR) mode, respectively. Benefiting from flexible picking-up ability, study migration at proteome level, demonstrating potential biological research insight.

Язык: Английский

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Microfluidic Biochips for Single‐Cell Isolation and Single‐Cell Analysis of Multiomics and Exosomes

Advanced Science, Год журнала: 2024, Номер 11(28)

Опубликована: Май 20, 2024

Abstract Single‐cell multiomic and exosome analyses are potent tools in various fields, such as cancer research, immunology, neuroscience, microbiology, drug development. They facilitate the in‐depth exploration of biological systems, providing insights into disease mechanisms aiding treatment. isolation, which is crucial for single‐cell analysis, ensures reliable cell isolation quality control further downstream analyses. Microfluidic chips small lightweight systems that efficient high‐throughput real‐time analysis on‐ or off‐chip. Therefore, most current technologies based on microfluidic technology. This review offers comprehensive guidance to researchers across different fields selection appropriate chip analysis. describes design principles, separation mechanisms, characteristics, cellular effects available isolation. Moreover, this highlights implications using technology subsequent analyses, including Finally, challenges future prospects outlined multiplex

Язык: Английский

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Top-down proteomics

Nature Reviews Methods Primers, Год журнала: 2024, Номер 4(1)

Опубликована: Июнь 13, 2024

Язык: Английский

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