Instrumentation at the Leading Edge of Proteomics

Analytical Chemistry, Год журнала: 2024, Номер 96(20), С. 7976 - 8010

Опубликована: Май 13, 2024

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTInstrumentation at the Leading Edge of ProteomicsTrenton M. Peters-ClarkeTrenton Peters-ClarkeDepartment Chemistry, University Wisconsin─Madison, Madison, Wisconsin 53706, United StatesDepartment Biomolecular StatesMore by Trenton Peters-ClarkeView Biographyhttps://orcid.org/0000-0002-9153-2525, Joshua J. CoonJoshua CoonDepartment StatesMorgridge Institute for Research, 53715, CoonView Biographyhttps://orcid.org/0000-0002-0004-8253, and Nicholas Riley*Nicholas RileyDepartment Washington, Seattle, Washington 98195, States*Email: [email protected]More RileyView Biographyhttps://orcid.org/0000-0002-1536-2966Cite this: Anal. Chem. 2024, 96, 20, 7976–8010Publication Date (Web):May 13, 2024Publication History Received6 October 2023Accepted19 April 2024Revised17 2024Published online13 May inissue 21 2024https://pubs.acs.org/doi/10.1021/acs.analchem.3c04497https://doi.org/10.1021/acs.analchem.3c04497review-articleACS PublicationsCopyright © 2024 American Chemical SocietyRequest reuse permissionsArticle Views2104Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are COUNTER-compliant sum full text article downloads since November 2008 (both PDF HTML) across all institutions individuals. These metrics regularly updated to reflect usage leading up last few days.Citations number other articles citing this article, calculated Crossref daily. Find more information about citation counts.The Altmetric Attention Score is a quantitative measure attention that research has received online. Clicking on donut icon will load page altmetric.com with additional details score social media presence given article. how calculated. Share Add toView InAdd Full Text ReferenceAdd Description ExportRISCitationCitation abstractCitation referencesMore Options onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Dissociation,Ions,Mass spectrometry,Peptides proteins,Proteomics Get e-Alerts

Язык: Английский

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Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июнь 8, 2023

Abstract The complexity of human physiology arises from well-orchestrated interactions between trillions single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding cell diversity, gene expression alone does not fully characterize phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics emerged a powerful tool for comprehensive protein analysis, including applications. However, challenges remain terms throughput and proteomic depth, order maximize biological impact by Spectrometry (scp-MS) workflows. This study leverages novel high-resolution, accurate mass (HRAM) instrument platform, consisting both an Orbitrap innovative HRAM Asymmetric Track Lossless (Astral) analyzer. Astral analyzer offers high sensitivity resolution through lossless ion transfer unique flight track design. We evaluate performance Thermo Scientific MS using Data-Independent Acquisition (DIA) assess proteome depth quantitative precision ultra-low input samples. Optimal DIA method parameters identified, we demonstrate ability cycle dynamics Human Embryonic Kidney (HEK293) cells, cancer heterogeneity primary Acute Myeloid Leukemia (AML) culture model.

Язык: Английский

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Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics

Analytical Chemistry, Год журнала: 2023, Номер 95(26), С. 9881 - 9891

Опубликована: Июнь 20, 2023

A linear ion trap (LIT) is an affordable, robust mass spectrometer that provides fast scanning speed and high sensitivity, where its primary disadvantage inferior accuracy compared to more commonly used time-of-flight or orbitrap (OT) analyzers. Previous efforts utilize the LIT for low-input proteomics analysis still rely on either built-in OTs collecting precursor data OT-based library generation. Here, we demonstrate potential versatility of as a stand-alone analyzer all spectrometry (MS) measurements, including To test this approach, first optimized acquisition methods performed library-free searches with without entrapment peptides evaluate both detection quantification accuracy. We then generated matrix-matched calibration curves estimate lower limit using only 10 ng starting material. While LIT-MS1 measurements provided poor quantitative accuracy, LIT-MS2 were quantitatively accurate down 0.5 column. Finally, suitable strategy spectral generation from material, which analyze single-cell samples by LIT-DIA LIT-based libraries few 40 cells.

Язык: Английский

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Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

Journal of the American Society for Mass Spectrometry, Год журнала: 2024, Номер 35(6), С. 1120 - 1127

Опубликована: Март 21, 2024

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption cell (SCP) human cells impeded by low sample loading capacity CZE, only allowing us to use less than 5% available peptide material each measurement. Here we present reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform solve issue, paving way SCP using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume connection via situ synthesis frit, followed packing C8 beads into form roughly 2 mm long SPME section. Peptides captured were eluted buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), dynamic pH junction-based SPME-CZE-MS enabled injection nearly 40% identified 257 ± 24 proteins 523 69 peptides (N = 2) Q-Exactive HF mass spectrometer when 0.25 ng commercial HeLa digest vial 0.1 injected. amount is equivalent protein cell. data indicate that ready cells.

Язык: Английский

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Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics

Communications Chemistry, Год журнала: 2024, Номер 7(1)

Опубликована: Апрель 10, 2024

Abstract Mapping the ligandability or potential druggability of all proteins in human proteome is a central goal mass spectrometry-based covalent chemoproteomics. Achieving this ambitious objective requires high throughput and coverage sample preparation liquid chromatography-tandem spectrometry analysis for hundreds to thousands reactive compounds chemical probes. Conducting chemoproteomic screens at scale benefits from technical innovations that achieve increased throughput. Here we realize vision by establishing silane-based cleavable linkers isotopically-labeled proteomics-tandem tag (sCIP-TMT) proteomic platform, which distinguished early pooling increases sCIP-TMT pairs custom click-compatible sCIP capture reagent readily functionalized yield with commercially available TMT reagents. Synthesis benchmarking 10-plex set reveal substantial decrease time together accuracy quantification. By screening focused four cysteine-reactive electrophiles, demonstrate utility target hunting, identifying 789 total liganded cysteines. Distinguished its compatibility established enrichment quantification protocols, expect will translate wide range applications.

Язык: Английский

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Proteome-scale tissue mapping using mass spectrometry based on label-free and multiplexed workflows

Molecular & Cellular Proteomics, Год журнала: 2024, Номер 23(11), С. 100841 - 100841

Опубликована: Сен. 20, 2024

Язык: Английский

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Trends in Mass Spectrometry-Based Single-Cell Proteomics

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Март 16, 2025

InfoMetrics Analytical ChemistryASAPArticle CiteCitationCitation and abstractCitation referencesMore citation options ShareShare onFacebookXWeChatLinkedInRedditEmailBlueskyJump toExpandCollapse ReviewMarch 16, 2025Trends in Mass Spectrometry-Based Single-Cell ProteomicsClick to copy article linkArticle link copied!Ximena Sanchez-AvilaXimena Sanchez-AvilaDepartment of Chemistry Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Ximena Sanchez-AvilaView BiographyRaphaela M. de OliveiraRaphaela OliveiraDepartment Raphaela OliveiraView BiographySiqi HuangSiqi HuangDepartment Siqi HuangView BiographyChao WangChao WangDepartment Chao WangView Biographyhttps://orcid.org/0009-0008-6197-2985Ryan T. Kelly*Ryan KellyDepartment States*Email: [email protected]More Ryan KellyView Biographyhttps://orcid.org/0000-0002-3339-4443Other Access OptionsAnalytical ChemistryCite this: Anal. Chem. 2025, XXXX, XXX, XXX-XXXClick citationCitation copied!https://pubs.acs.org/doi/10.1021/acs.analchem.5c00661https://doi.org/10.1021/acs.analchem.5c00661Published March 2025 Publication History Received 28 January 2025Accepted February 2025Revised 23 2025Published online 16 2025review-article© American Chemical SocietyRequest reuse permissionsACS Publications© SocietySubjectswhat are subjects Article automatically applied from the ACS Subject Taxonomy describe scientific concepts themes article. Cells Isolation spectrometry Peptides proteins Sample preparation Note: In lieu an abstract, this is article's first page. Read To access article, please review available below. Get instant Purchase for 48 hours. Check out below using your ID or as a guest. Restore my guest Recommended through Your Institution You may have institution. institution does not content. Add change let them know you'd like include access. Through Recommend Name Loading Institutional Login Options... Change Explore subscriptions institutions Log with if you previously purchased it member benefits. hours $48.00 cart Checkout Cited By Click section linkSection copied!This has yet been cited other publications.Download PDF e-AlertsGet e-AlertsAnalytical copied!https://doi.org/10.1021/acs.analchem.5c00661Published 2025© permissionsArticle Views6Altmetric-Citations-Learn about these metrics closeArticle Views COUNTER-compliant sum full text downloads since November 2008 (both HTML) across all individuals. These regularly updated reflect usage leading up last few days.Citations number articles citing calculated Crossref daily. Find more information counts.The Altmetric Attention Score quantitative measure attention that research received online. Clicking on donut icon will load page at altmetric.com additional details score social media presence given how calculated.Recommended Articles

Язык: Английский

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Challenges and Opportunities for Single-cell Computational Proteomics

Molecular & Cellular Proteomics, Год журнала: 2023, Номер 22(4), С. 100518 - 100518

Опубликована: Фев. 23, 2023

Single-cell proteomics is growing rapidly and has made several technological advancements. As most research been focused on improving instrumentation sample preparation methods, very little attention given to algorithms responsible for identifying quantifying proteins. Given the inherent difference between bulk data single-cell data, it necessary realize that current being employed were designed have underlying assumptions may not hold true data. In order develop optimize we need characterize differences assess how perform Here, present a review of peptides We will give each type algorithm works, relies on, performs possible optimizations solutions could be used address in

Язык: Английский

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Quantitative Label-Free Single-Cell Proteomics on the Orbitrap Astral MS

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

Опубликована: Июль 31, 2024

Abstract Single-cell proteomics by mass spectrometry (scp-MS) holds the potential to provide unprecedented insights into molecular features directly linked cellular phenotype, while deconvoluting complex organisms their basic building blocks. Tailored sample preparation that maximizes extracted amount of material is introduced spectrometer has rapidly propelled field forward. However, measured signal still at lower edge detection approaching sensitivity boundary current instrumentation. Here, we investigate capacity enhanced Orbitrap Astral facilitate deeper proteome profiles from low-input single-cell samples. We carry out a comprehensive data acquisition method survey pinpoint which parameters most sensitivity. Furthermore, explore quantitative accuracy obtained measurements ensure abundances are in line with expected ground truth values. culminate our technical exploration generating small datasets two cultured cell lines and primary bone marrow sample, showcase obtainable coverage differences different source materials. Finally, as proof concept protein covariation how information on known complexes captured inherently scp-MS data.

Язык: Английский

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Data Independent Acquisition Approaches for Single Cell Proteomics

Authorea (Authorea), Год журнала: 2024, Номер unknown

Опубликована: Авг. 1, 2024

Single-cell proteomics (SCP) aims to characterize the proteome of individual cells, providing insights into complex biological systems. It reveals subtle differences in distinct cellular populations that bulk analysis might overlook, which is essential for understanding disease mechanisms and developing targeted therapies. Mass spectrometry (MS) methods SCP allow identification quantification thousands proteins from cells this review highlights role data-independent acquisition MS (DIA-MS) SCP. One major hurdle limited material single-cell samples, but DIA-based techniques offer multiple potential solutions their analysis. Utilizing wide precursor isolation windows fragment peptides simultaneously, improve sensitivity, quantitative accuracy, reproducibility at a cost data complexity. DIA can also be combined with sample multiplexing increase throughput, currently key limitation Challenges remain interpreting multiplexed experiments, particularly regards isobaric tagging methods. Even still, we believe approaches will play our systems biology.

Язык: Английский

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