HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics DOI
Yiran Liang, Thy Truong, Aubrianna J. Saxton

et al.

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(20), P. 8020 - 8027

Published: May 11, 2023

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods been separately applied to SCP increase through multiplexing. Here we combined both forms of achieve multiplicative scaling for higher throughput. Two-plex stable isotope amino acids cell culture (SILAC) isobaric tandem tag (TMT) enabled up 28 single cells be analyzed liquid chromatography–mass spectrometry (LC–MS) analysis, addition carrier, reference, negative control channels. A custom nested nanowell chip was used nanoliter sample processing minimize losses. Using 145-min total LC–MS cycle time, ∼280 were per day. This could increased ∼700 samples day with high-duty-cycle multicolumn LC system producing same active gradient. The efficiency achievable proteome coverage characterized multiple analysis conditions.

Language: Английский

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles DOI Open Access
Paul Dowling, Stephen Gargan, Dieter Swandulla

et al.

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(3), P. 2415 - 2415

Published: Jan. 26, 2023

The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia the elderly, which is characterized by severe muscular atrophy that can considerably lessen overall quality life at old age. Mass-spectrometry-based proteomic surveys senescent human muscles, as well animal models sarcopenia, have decisively improved our understanding molecular cellular consequences fiber-type shifting during aging. This review outlines spectrometric identification proteome-wide changes atrophying focus on proteins potential markers distribution patterns. observed trend fast-to-slow transitions individual muscles aging process most likely linked to preferential susceptibility fast-twitching fibers atrophy. Studies models, including mostly aged rodent confirmed shifting. analysis fast versus slow isoforms key proteins, such myosin heavy chains, light actins, troponins tropomyosins, suggests them suitable bioanalytical tools

Language: Английский

Citations

46

A review of the current state of single-cell proteomics and future perspective DOI Creative Commons
Rushdy Ahmad, Bogdan Budnik

Analytical and Bioanalytical Chemistry, Journal Year: 2023, Volume and Issue: 415(28), P. 6889 - 6899

Published: June 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.

Language: Английский

Citations

44

Pick-up single-cell proteomic analysis for quantifying up to 3000 proteins in a Mammalian cell DOI Creative Commons
Yu Wang,

Zhi-Ying Guan,

Shao-Wen Shi

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Feb. 10, 2024

Abstract The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell still insufficient for practical applications. Here, we develop a pick-up (PiSPA) workflow to achieve deep capable quantifying up 3000 groups in mammalian using label-free quantitative method. PiSPA specially established samples mainly based on nanoliter-scale microfluidic liquid handling robot, achieving capture, pretreatment and injection under operation strategy. Using this customized with remarkable improvement identification, 2449–3500, 2278–3257 1621–2904 are quantified single A549 cells ( n = 37), HeLa 44) U2OS 27) DIA (MBR) mode, respectively. Benefiting from flexible picking-up ability, study migration at proteome level, demonstrating potential biological research insight.

Language: Английский

Citations

27

Top-down proteomics DOI
David S. Roberts, Joseph A. Loo, Yury O. Tsybin

et al.

Nature Reviews Methods Primers, Journal Year: 2024, Volume and Issue: 4(1)

Published: June 13, 2024

Language: Английский

Citations

20

High-resolution spatially resolved proteomics of complex tissues based on microfluidics and transfer learning DOI Creative Commons
Beiyu Hu,

Ruiqiao He,

Kun Pang

et al.

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Despite recent advances in imaging- and antibody-based methods, achieving in-depth, high-resolution protein mapping across entire tissues remains a significant challenge spatial proteomics. Here, we present parallel-flow projection transfer learning omics data (PLATO), an integrated framework combining microfluidics with deep to enable of thousands proteins whole tissue sections. We validated the PLATO by profiling proteome mouse cerebellum, identifying 2,564 groups single run. then applied rat villus human breast cancer samples, resolution 25 μm uncovering proteomic dynamics associated disease states. This approach revealed spatially distinct tumor subtypes, identified key dysregulated proteins, provided novel insights into complexity microenvironment. believe that represents transformative platform for exploring regulation its interplay genetic environmental factors.

Language: Английский

Citations

4

MYC multimers shield stalled replication forks from RNA polymerase DOI
Daniel Solvie, Apoorva Baluapuri, Leonie Uhl

et al.

Nature, Journal Year: 2022, Volume and Issue: 612(7938), P. 148 - 155

Published: Nov. 23, 2022

Language: Английский

Citations

58

Scaling Up Single-Cell Proteomics DOI Creative Commons
Nikolai Slavov

Molecular & Cellular Proteomics, Journal Year: 2021, Volume and Issue: 21(1), P. 100179 - 100179

Published: Nov. 20, 2021

Single-cell tandem MS has enabled analyzing hundreds of single cells per day and quantifying thousands proteins across the cells. The broad dissemination these capabilities can empower dissection pathophysiological mechanisms in heterogeneous tissues. Key requirements for achieving this goal include robust protocols performed on widely accessible hardware, quality controls, community standards, automated data analysis pipelines that pinpoint analytical problems facilitate their timely resolution. Toward meeting requirements, perspective outlines both existing resources outstanding opportunities, such as parallelization, catalyzing wide quantitative single-cell proteomics be scaled up to tens Indeed, simultaneous parallelization peptides is a promising approach multiplicative increase speed performing deep proteomics. ready begin virtuous cycle increased adoption fueling development more technology turn drive broader adoption, scientific discoveries, clinical applications.

Language: Английский

Citations

56

Recent advances in isobaric labeling and applications in quantitative proteomics DOI

Michael K. Sivanich,

Ting‐Jia Gu, Dylan Nicholas Tabang

et al.

PROTEOMICS, Journal Year: 2022, Volume and Issue: 22(19-20)

Published: June 10, 2022

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass (LC-MS) can be used to determine abundances proteins and peptides in complex biological samples. Several methods have been developed adapted for accurate quantification based on chemical isotopic labeling. Among various labeling techniques, isobaric tagging approaches rely analysis from MS2-based rather than MS1-based quantification. In this review, we will provide an overview several tags along with some recent developments including complementary ion tags, improvements sensitive quantitation analytes lower abundance, strategies increase multiplexing capabilities, targeted strategies. We also discuss limitations alleviate these restrictions through bioinformatic tools data acquisition methods. This review highlight applications biomarker discovery validation, thermal proteome profiling, cross-linking structural investigations, single-cell analysis, top-down proteomics, different molecules neuropeptides, glycans, metabolites, lipids, while providing considerations evaluations each application.

Language: Английский

Citations

56

High-end ion mobility mass spectrometry: A current review of analytical capacity in omics applications and structural investigations DOI Creative Commons
Daniel G. Delafield, Gaoyuan Lu, Cameron J. Kaminsky

et al.

TrAC Trends in Analytical Chemistry, Journal Year: 2022, Volume and Issue: 157, P. 116761 - 116761

Published: Aug. 24, 2022

Language: Английский

Citations

52

Critical considerations in N-glycoproteomics DOI
The Huong Chau, Anastasia Chernykh, Rebeca Kawahara

et al.

Current Opinion in Chemical Biology, Journal Year: 2023, Volume and Issue: 73, P. 102272 - 102272

Published: Feb. 7, 2023

Language: Английский

Citations

38