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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Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

ACS Measurement Science Au, Год журнала: 2024, Номер 4(4), С. 338 - 417

Опубликована: Июнь 4, 2024

Proteomics is the large scale study of protein structure and function from biological systems through identification quantification."Shotgun proteomics" or "bottom-up prevailing strategy, in which proteins are hydrolyzed into peptides that analyzed by mass spectrometry.Proteomics studies can be applied to diverse ranging simple proteoforms, protein-protein interactions, structural alterations, absolute relative quantification, post-translational modifications, stability.To enable this range different experiments, there strategies for proteome analysis.The nuances how proteomic workflows differ may challenging understand new practitioners.Here, we provide a comprehensive overview proteomics methods.We cover biochemistry basics extraction interpretation orthogonal validation.We expect Review will serve as handbook researchers who field bottom-up proteomics.

Язык: Английский

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Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

Journal of the American Society for Mass Spectrometry, Год журнала: 2023, Номер 34(10), С. 2374 - 2380

Опубликована: Авг. 18, 2023

Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables, and reagents for sample processing separations, which has limited the accessibility SCP to a small number specialized laboratories. Commercial platforms have become available cell isolation preparation, but high cost these technical expertise required their operation place them out reach many interested Here, we assessed new HP D100 Single Cell Dispenser label-free SCP. The low-cost instrument proved highly accurate reproducible dispensing in range from 200 nL 2 μL. We used isolate prepare single cells within 384-well PCR plates. When well plates were immediately centrifuged following again after reagent dispensing, found ∼97% wells identified software as containing indeed provided expected cell. This commercial dispenser combined with one-step provides very rapid easy-to-use workflow no reduction relative nanowell-based workflow, also facilitate autosampling unmodified instrumentation. samples analyzed using home-packed 30 μm i.d. nanoLC columns commercially 50 columns. resulted ∼35% fewer proteins. However, plate-based preparation platform, presented fully relatively alternative separation, should greatly broaden

Язык: Английский

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Proteomics of the heart

Physiological Reviews, Год журнала: 2024, Номер 104(3), С. 931 - 982

Опубликована: Фев. 1, 2024

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at molecular level. Proteomics provides biologists with snapshot of context-dependent and proteoform expression, structural conformations, dynamic turnover, protein-protein interactions. Cardiac can offer broader deeper understanding the mechanisms that underscore cardiovascular disease, it foundational to development future therapeutic interventions. This review encapsulates evolution, current technologies, perspectives proteomic-based mass spectrometry as applies study heart. Key technological advancements have allowed researchers proteomes single-cell level employ robot-assisted automation systems for enhanced sample preparation techniques, increase fidelity spectrometers has unambiguous numerous posttranslational modifications. Animal models ranging from early animal experiments heart failure preserved ejection fraction, provided tools challenging organ laboratory. Further will pave way implementation even closer within clinical setting, allowing not only scientists but also patients benefit an interplay relates cardiac disease physiology.

Язык: Английский

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Single cell proteomics by mass spectrometry reveals deep epigenetic insight into the actions of an orphan histone deacetylase inhibitor

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Янв. 6, 2024

Abstract Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone thought look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur single cell resolution across population remains elusive. While recent advances sequencing technology have allowed between 1 3 histone post-translational be analyzed each cell, over twenty separate chemical PTMs are known exist, allowing thousands possible combinations. Single proteomics by mass spectrometry (SCP) is an emerging hundreds proteins can directly quantified typical cells. As detected SCP heavily biased toward highest abundance, chromatin attractive target for analysis. To this end, I applied analysis cancer cells treated with mocetinostat, class specific deacetylase inhibitor. find that 16 confidently identified localized high site specificity In addition, abundance allows higher throughput methods utilized than previously described. quantitative accuracy suffers when analyzing more 700 per day, 9 measured even 3,500 10-fold greater any previous report. unbiased global approach herein identifies uncharacterized response drug through S100-A8/S100-A9 protein complex partners. This observed 1,000 study, regardless relative method utilized. limitations exist described herein, current technologies easily improve upon results presented here allow comprehensive performed lab. All raw processed data study made publicly available ProteomeXchange/MASSIVE repository as MSV000093434 graphic

Язык: Английский

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A High-Sensitivity Low-Nanoflow LC-MS Configuration for High-Throughput Sample-Limited Proteomics

Analytical Chemistry, Год журнала: 2023, Номер 95(51), С. 18673 - 18678

Опубликована: Дек. 13, 2023

This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell (SCP). Conditions were optimized on a 50 μm internal diameter (I.D.) column operated at 100 nL/min in direct injection workflow to balance method sensitivity sample throughput from 24 72 samples/day. Multiple data acquisition strategies also evaluated proteome coverage, data-dependent (DDA), wide-window (WWA), data-independent (WW-DIA). Analyzing 250 pg HeLa digest with 10-min LC gradient (72 samples/day) provided >900, >1,800, >3,000 protein group identifications DDA, WWA, WW-DIA, respectively. Total cycle time was further reduced 20 14.4 min (100 by employing trap-and-elute workflow, enabling 70% mass spectrometer utilization. The applied library-free DIA analysis samples, yielding >1,700 groups identified. In conclusion, this study provides high-sensitivity, configuration proteomics.

Язык: Английский

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Single‐Cell Patch‐Clamp/Proteomics of Human Alzheimer's Disease iPSC‐Derived Excitatory Neurons Versus Isogenic Wild‐Type Controls Suggests Novel Causation and Therapeutic Targets

Advanced Science, Год журнала: 2024, Номер 11(29)

Опубликована: Май 21, 2024

Abstract Standard single‐cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to physiology. Here, a scPatch‐Clamp/Proteomics platform is developed on single neurons generated hiPSCs bearing an Alzheimer's (AD) genetic mutation and compares them isogenic wild‐type controls. This approach provides both current voltage electrophysiological data plus detailed information single‐cells. With this new method, the authors are able observe hyperelectrical activity in AD hiPSC‐neurons, similar that observed human brain, correlate it ≈1400 proteins detected at neuron level. Using linear regression mediation analyses explore relationship between abundance individual neuron's mutational status, yields therapeutic targets excitatory not attainable by traditional methods. combined patch‐proteomics technique creates proteogenetic‐therapeutic strategy genotypic alterations physiology with protein expression

Язык: Английский

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The Future of Proteomics is Up in the Air: Can Ion Mobility Replace Liquid Chromatography for High Throughput Proteomics?

Journal of Proteome Research, Год журнала: 2024, Номер 23(6), С. 1871 - 1882

Опубликована: Май 7, 2024

The coevolution of liquid chromatography (LC) with mass spectrometry (MS) has shaped contemporary proteomics. LC hyphenated to MS now enables quantification more than 10,000 proteins in a single injection, number that likely represents most specific human cells or tissues. Separations by ion mobility (IMS) have recently emerged complement and further improve the depth Given theoretical advantages speed robustness IMS comparison LC, we envision ongoing improvements paired may eventually make obsolete, especially when combined targeted simplified analyses, such as rapid clinical proteomics analysis defined biomarker panels. In this perspective, describe need for faster might drive transition, current state direct infusion proteomics, discuss some technical challenges must be overcome fully complete transition entirely gas phase

Язык: Английский

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Deep topographic proteomics of a human brain tumour

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

Опубликована: Ноя. 24, 2023

Abstract The spatial organisation of cellular protein expression profiles within tissue determines function and is key to understanding disease pathology. To define molecular phenotypes in the context tissue, there a need for unbiased, quantitative technology capable mapping proteomes structures. Here, we present workflow spatially-resolved, proteomics that generates maps abundance across slices derived from human atypical teratoid-rhabdoid tumour at three resolutions, highest being 40 µm, reveal distinct patterns thousands proteins. We employ spatially-aware algorithms do not require prior knowledge fine structure detect proteins pathways with correlate heterogeneity features such as extracellular matrix or proximity blood vessels. identify PYGL, ASPH CD45 markers boundary immune response-driven, spatially-organised networks matrix. Overall, demonstrate deep proteo-phenotyping heterogeneity, re-define biology pathology level.

Язык: Английский

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A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

Journal of the American Society for Mass Spectrometry, Год журнала: 2023, Номер 34(9), С. 1858 - 1867

Опубликована: Июль 18, 2023

Skeletal muscle is a major regulatory tissue of whole-body metabolism and composed diverse mixture cell (fiber) types. Aging several diseases differentially affect the various fiber types, therefore, investigating changes in proteome fiber-type specific manner essential. Recent breakthroughs isolated single proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow laborious, requiring 2 h mass spectrometry time per fiber; 50 fibers would take approximately 4 days analyze. Thus, capture high variability both within between individuals requires advancements throughput proteomics. Here we use method enable quantification proteomes 15 min total instrument time. As proof concept, present data from 53 skeletal obtained two healthy analyzed 13.25 h. Adapting analysis techniques integrate data, can reliably separate type 1 2A Ninety-four proteins were statistically different clusters indicating alteration involved fatty acid oxidation, oxidative phosphorylation, structure contractile function. Our results indicate that this significantly faster than prior methods collection sample preparation while maintaining sufficient depth. We anticipate assay will future studies across hundreds individuals, which has not been possible previously due limitations throughput.

Язык: Английский

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