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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Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics

Nature Methods, Год журнала: 2023, Номер 20(5), С. 714 - 722

Опубликована: Апрель 3, 2023

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth protein quantification, especially for proteins modifications biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands peptides across single cells (thus data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus proteome depth. These strategies increased sensitivity, completeness coverage over twofold. The gains enabled quantifying variation in untreated lipopolysaccharide-treated primary macrophages. Within each condition, covaried within functional sets, including phagosome maturation proton transport, similarly both treatment conditions. This covariation is coupled phenotypic variability endocytic activity. also proteolytic products, suggesting a gradient cathepsin activities condition. freely available widely applicable, interest without sacrificing coverage. Support at http://scp.slavovlab.net/pSCoPE .

Язык: Английский

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Sampling the proteome by emerging single-molecule and mass spectrometry methods

Nature Methods, Год журнала: 2023, Номер 20(3), С. 339 - 346

Опубликована: Март 1, 2023

Язык: Английский

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Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel

Molecular Systems Biology, Год журнала: 2023, Номер 19(9)

Опубликована: Авг. 21, 2023

Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited proteomic depth, throughput, robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated complete dimethyl labeling bulk or single-cell samples, without losing depth. Lys-N digestion enables five-plex quantification MS1 MS2 level. Because channels are quantitatively isolated from each other, mDIA accommodates reference channel that does not interfere with target channels. Our algorithm RefQuant takes advantage this confidently quantifies twice as many per single cell compared our previous work (Brunner et al, PMID 35226415), while allows routine analysis 80 cells day. Finally, combined spatial increase throughput Deep Visual Proteomics seven-fold for microdissection four-fold MS analysis. Applying primary cutaneous melanoma, discovered signatures within distinct tumor microenvironments, showcasing its potential precision oncology.

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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