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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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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Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications

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

Опубликована: Июль 8, 2024

Abstract The recent technological and computational advances in mass spectrometry-based single-cell proteomics have pushed the boundaries of sensitivity throughput. However, reproducible quantification thousands proteins within a single cell remains challenging. To address some those limitations, we present dedicated sample preparation chip, proteoCHIP EVO 96 that directly interfaces with Evosep One. This, combination Bruker timsTOF demonstrates double identifications without manual handling newest generation Ultra identifies up to 4000 an average 3500 protein groups per HEK-293T carrier or match-between runs. Our workflow spans 4 orders magnitude, over 50 E3 ubiquitin-protein ligases, profiles key regulatory upon small molecule stimulation. This study 96-based provides sufficient proteome depth complex biology beyond cell-type classifications.

Язык: Английский

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Micropillar arrays, wide window acquisition and AI-based data analysis improve comprehensiveness in multiple proteomic applications

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

Опубликована: Фев. 3, 2024

Comprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited coverage dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition the AI-based CHIMERYS search engine achieve excellent comprehensiveness for bulk affinity purification mass spectrometry single cell proteomics. Our data show that µPACs identify ≤50% more peptides ≤24% proteins, while offering improved throughput, which critical large (clinical) proteomics studies. Combining wide precursor isolation widths of m/z 4-12 identified +51-74% +59-150% proteins peptides, respectively, cell, co-immunoprecipitation, multi-species samples over a conventional workflow at well-controlled false discovery rates. The further offers precision, CVs <7% low input samples, accuracy, deviations <10% expected fold changes regular abundance two-proteome mixes. Compared workflow, our entire optimized platform discovered 92% potential interactors protein-protein interaction study on chromatin remodeler Smarca5/Snf2h. These include previously described Smarca5 binding partners undescribed ones including Arid1a, another key roles neurodevelopmental malignant disorders.

Язык: Английский

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Enhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics

Nature Methods, Год журнала: 2025, Номер unknown

Опубликована: Янв. 16, 2025

Single-cell proteomics (SCP) promises to revolutionize biomedicine by providing an unparalleled view of the proteome in individual cells. Here, we present a high-sensitivity SCP workflow named Chip-Tip, identifying >5,000 proteins HeLa It also facilitated direct detection post-translational modifications single cells, making need for specific modification-enrichment unnecessary. Our study demonstrates feasibility processing up 120 label-free samples per day. An optimized tissue dissociation buffer enabled effective single-cell disaggregation drug-treated cancer cell spheroids, refining overall analysis. Analyzing nondirected human-induced pluripotent stem differentiation, consistently quantified markers OCT4 and SOX2 cells lineage such as GATA4 (endoderm), HAND1 (mesoderm) MAP2 (ectoderm) different embryoid body sets benchmark sensitivity throughput, with broad applications basic biology identification type-specific therapeutic targets.

Язык: Английский

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Challenging the Astral mass analyzer to quantify up to 5,300 proteins per single cell at unseen accuracy to uncover cellular heterogeneity

Nature Methods, Год журнала: 2025, Номер unknown

Опубликована: Янв. 16, 2025

Язык: Английский

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Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform

Analytical Chemistry, Год журнала: 2022, Номер 94(15), С. 6017 - 6025

Опубликована: Апрель 6, 2022

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due improved ionization efficiency, but the time required for sample loading, column washing, regeneration in these systems can lead low measurement throughput inefficient utilization mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically label-free SCP using two parallel subsystems multiplex online desalting, analysis, regeneration. integration MS1-based feature matching increased proteome coverage when short LC gradients were used. high-throughput was reproducible between columns, 4% difference median peptide abundance CV 18% across 100 replicate analyses single-cell-sized standard. An average 621, 774, 952, 1622 protein groups identified total analysis times 7, 10, 15, 30 min, corresponding 206, 144, 96, 48 samples per day, respectively. When applied single HeLa cells, nearly 1000 cell min cycles 660 15 cycles. We explored possibility measuring cancer therapeutic targets pilot study comparing K562 Jurkat leukemia lines. This work demonstrates feasibility single-cell proteomics.

Язык: Английский

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Label‐free single cell proteomics utilizing ultrafast LC and MS instrumentation: A valuable complementary technique to multiplexing

PROTEOMICS, Год журнала: 2023, Номер 23(13-14)

Опубликована: Фев. 19, 2023

The ability to map a proteomic fingerprint transcriptomic data would master the understanding of how gene expression translates into actual phenotype. In contrast nucleic acid sequencing, in vitro protein amplification is impossible and no single cell workflow has been established as gold standard yet. Advances microfluidic sample preparation, multi-dimensional separation, sophisticated acquisition strategies, intelligent analysis algorithms have resulted major improvements successfully analyze such tiny amounts with steadily boosted performance. However, among broad variation published approaches, it commonly accepted that highest possible sensitivity, robustness, throughput are still most urgent needs for field. While many labs focused on multiplexing achieve these goals, label-free SCP highly promising strategy well whenever high dynamic range unbiased accurate quantification needed. We here focus recent advances single-cell mass spectrometry workflows try guide our readers choose best method or combinations methods their specific applications. further highlight which techniques propitious future applications but also limitations we foresee

Язык: Английский

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Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics

Journal of the American Society for Mass Spectrometry, Год журнала: 2023, Номер 34(8), С. 1701 - 1707

Опубликована: Июль 6, 2023

Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive lead to long sample-to-answer times. Here we report sample method that achieves cell lysis, protein denaturation, digestion 1 h using commercially available high-temperature-stabilized proteases single reagent step. Four different one-step compositions were evaluated, the mixture providing highest proteome coverage was compared previously employed multistep workflow. The increases relative previous while minimizing input possibility of human error. We also recovery between used microfabricated glass nanowell chips injection-molded polypropylene found provided improved coverage. Combined, substrates enabled identification an average nearly 2400 proteins per standard data-dependent Orbitrap mass spectrometers. advances greatly simplify broaden accessibility no compromise terms

Язык: Английский

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