High-throughput and scalable single cell proteomics identifies over 5000 proteins per cell

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

Опубликована: Ноя. 28, 2023

Abstract The emergence of mass spectrometry (MS)-based single-cell proteomics (SCP) promise to revolutionize the study cellular biology and biomedicine by providing an unparalleled view proteome in individual cells. Despite its groundbreaking potential, SCP is nascent faces challenges including limited sequence depth, throughput, reproducibility, which have constrained broader utility. This introduces key methodological advances, considerably improve sensitivity, coverage dependability protein identification from single We developed almost lossless workflow encompassing sample preparation MS analysis, doubling number identified proteins roughly 2000 over 5000 HeLa A comprehensive evaluation analytical software tools, alongside strict false discovery rate (FDR) controls solidified reliability our results. These enhancements also facilitated direct detection post-translational modifications (PTMs) cells, negating need for enrichment thereby simplifying process. Although throughput remains a challenge, demonstrates feasibility processing up 80 label-free samples per day. Moreover, optimized tissue dissociation buffer enabled effective cell disaggregation drug-treated cancer spheroids, refining overall proteomic analysis. Our sets new benchmark sensitivity with broad applications ranging development disease progression type-specific markers therapeutic targets.

Язык: Английский

---

Dynamics of Single-Cell Protein Covariation during Epithelial–Mesenchymal Transition

Journal of Proteome Research, Год журнала: 2024, Номер unknown

Опубликована: Апрель 25, 2024

Physiological processes, such as the epithelial–mesenchymal transition (EMT), are mediated by changes in protein interactions. These may be better reflected covariation within a cellular cluster than temporal dynamics of cluster-average abundance. To explore this possibility, we quantified proteins single human cells undergoing EMT. Covariation analysis data revealed that functionally coherent clusters dynamically changed their protein–protein correlations without concomitant were monotonic time and delineated modules functioning actin cytoskeleton organization, energy metabolism, transport. defined same point and, thus, reflect biological regulation masked dynamics. Thus, correlation across offers window into during physiological transitions.

Язык: Английский

---

Multicolumn Nanoflow Liquid Chromatography with Accelerated Offline Gradient Generation for Robust and Sensitive Single-Cell Proteome Profiling

Analytical Chemistry, Год журнала: 2024, Номер 96(26), С. 10534 - 10542

Опубликована: Июнь 25, 2024

Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion support multiple analytical columns, trap column interfaces all columns reduce required maintenance simplify troubleshooting. A degree of parallelization is possible, as one sample undergoes separation while the next plus its corresponding mobile phase transferred into storage loop third loaded loop. Selective elution from prevents salts hydrophobic species entering column, thus greatly enhancing lifetime robustness. With design, can be analyzed fast every 20 min at flow rate just 40 nL/min close 100% MS utilization time continuously long several months without replacement. We utilized analyze proteomes cells myeloma cell line upon treatment immunomodulatory imide drug lenalidomide.

Язык: Английский

---

Cell Storage Conditions Impact Single-Cell Proteomic Landscapes

Journal of Proteome Research, Год журнала: 2025, Номер unknown

Опубликована: Янв. 24, 2025

Single cell transcriptomics (SCT) has revolutionized our understanding of cellular heterogeneity, yet the emergence single proteomics (SCP) promises a more functional view dynamics. A challenge is that not all mass spectrometry facilities can perform SCP, and laboratories have access to sorting equipment required for which together motivate an interest in sending bulk samples through mail SCP analysis. Shipping requires storage, unknown effect on results. This study investigates impact storage conditions proteomic landscape at level, utilizing Data-Independent Acquisition (DIA) coupled with Parallel Accumulation Serial Fragmentation (diaPASEF). Three were compared 293T cells: (1) 37 °C (control), (2) 4 overnight, (3) −196 followed by liquid nitrogen preservation. Both cold frozen induced significant alterations diameter, elongation, proteome composition. By elucidating how alter morphology profiles, this contributes foundational technical information about sample preparation data quality.

Язык: Английский

---

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

---

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

---

Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry

Journal of Proteome Research, Год журнала: 2024, Номер unknown

Опубликована: Сен. 26, 2024

Separation in single-cell mass spectrometry (MS) improves molecular coverage and quantification; however, it also elongates measurements, thus limiting analytical throughput to study large populations of cells. Here, we advance the speed bottom-up proteomics by capillary electrophoresis (CE) high-resolution for proteomics. We adjust applied potential readily control duration electrophoresis. On HeLa proteome standard, shorter separation times curbed detection using data-dependent acquisition (DDA) but not data-independent (DIA) on an Orbitrap analyzer. This DIA method identified 1161 proteins vs 401 reference DDA within a 15 min effective from single HeLa-cell-equivalent (∼200 pg) digests. Label-free quantification found these exclusively DIA-identified lower domain concentration range, revealing sensitivity improvement. The approach significantly advanced reproducibility quantification, where ∼76% DIA-quantified had <20% coefficient variation ∼43% DDA. As proof principle, allowed us quantify 1242 subcellular niches single, neural-tissue fated cell live

Язык: Английский

---

Formaldehyde Fixation Helps Preserve the Proteome State during Single-Cell Proteomics Sample Processing and Analysis

Journal of Proteome Research, Год журнала: 2025, Номер unknown

Опубликована: Фев. 3, 2025

Mass spectrometry-based single-cell proteomics (SCP) is gaining momentum but remains limited to a few laboratories due the high costs and specialized expertise required. The ability send samples core facilities would benefit nonspecialist popularize SCP for biological applications. However, no methods have been tested in "freeze" proteome state while maintaining cell integrity transfer between or prolonged sorting using fluorescence-activated (FACS). This study evaluates whether short-term formaldehyde (FA) fixation can maintain states. We demonstrate that FA does not substantially affect protein recovery, even without heating strong detergents, maintains analytical depth compared with classical workflows. Fixation also preserves drug-induced specific perturbations of abundance during sample preparation analysis. Our findings suggest facilitate by enabling shipping sorting, potentially democratizing access technology expanding its application research, thereby accelerating discoveries biology personalized medicine.

Язык: Английский

---

IS-SCP: enhanced single-cell proteomics using an in situ simplified strategy

The Analyst, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

A workflow for single-cell proteomic analysis was developed, named in situ simplified proteomics (IS-SCP), based on a comprehensive evaluation of the reagents, reaction conditions, and reproducibility analysis.

Язык: Английский

---

Global analysis of protein turnover dynamics in single cells

Cell, Год журнала: 2025, Номер unknown

Опубликована: Март 1, 2025

Single-cell proteomics (SCPs) has advanced significantly, yet it remains largely unidimensional, focusing primarily on protein abundances. In this study, we employed a pulsed stable isotope labeling by amino acids in cell culture (pSILAC) approach to simultaneously analyze abundance and turnover single cells (SC-pSILAC). Using state-of-the-art SCP workflow, demonstrated that two SILAC labels are detectable from ∼4,000 proteins HeLa recapitulating known biology. We performed large-scale time-series SC-pSILAC analysis of undirected differentiation human induced pluripotent stem (iPSCs) encompassing 6 sampling times over 2 months analyzed >1,000 cells. Protein dynamics highlighted differentiation-specific co-regulation complexes with core histone turnover, discriminating dividing non-dividing Lastly, correlating diameter the individual showed histones some cell-cycle do not scale size. The method provides multidimensional view single-cell

Язык: Английский

---