Advancements in Global Phosphoproteomics Profiling: Overcoming Challenges in Sensitivity and Quantification DOI Creative Commons
Gul Muneer,

Ciao‐Syuan Chen,

Yu‐Ju Chen

и другие.

PROTEOMICS, Год журнала: 2024, Номер unknown

Опубликована: Дек. 18, 2024

Protein phosphorylation introduces post-genomic diversity to proteins, which plays a crucial role in various cellular activities. Elucidation of system-wide signaling cascades requires high-performance tools for precise identification and quantification dynamics site-specific events. Recent advances phosphoproteomic technologies have enabled the comprehensive mapping dynamic landscape, has opened new avenues exploring cell type-specific functional networks underlying functions clinical phenotypes. Here, we provide an overview basics challenges phosphoproteomics, as well technological evolution current state-of-the-art global quantitative phosphoproteomics methodologies. With specific focus on highly sensitive platforms, summarize recent trends innovations miniaturized sample preparation strategies micro-to-nanoscale single-cell profiling, data-independent acquisition mass spectrometry (DIA-MS) enhanced coverage, pipelines deep disease biology. Each aspect analysis presents unique opportunities improvement innovation. We specifically highlight evolving that enable profiling from low-input samples. Finally, discuss persistent technologies, including feasibility nanoscale future outlooks biomedical applications.

Язык: Английский

An accessible workflow for high-sensitivity proteomics using parallel accumulation–serial fragmentation (PASEF) DOI
Patricia Skowronek, Georg Wallmann, Maria Wahle

и другие.

Nature Protocols, Год журнала: 2025, Номер unknown

Опубликована: Янв. 17, 2025

Язык: Английский

Процитировано

1
A network-based approach to overcome BCR::ABL1-independent resistance in chronic myeloid leukemia DOI Creative Commons
Valeria Bica, Veronica Venafra, Giorgia Massacci

и другие.

Cell Communication and Signaling, Год журнала: 2025, Номер 23(1)

Опубликована: Апрель 10, 2025

About 40% of relapsed or non-responder tumors exhibit therapeutic resistance in the absence a clear genetic cause, suggesting pivotal role intracellular communication. A deeper understanding signaling pathways rewiring occurring resistant cells is crucial to propose alternative effective strategies for cancer patients. To achieve this goal, we developed novel multi-step strategy, which integrates high sensitive mass spectrometry-based phosphoproteomics with network-based analysis. This strategy builds context-specific networks recapitulating upon drug treatment therapy-resistant and cells. We applied elucidate BCR::ABL1-independent mechanisms that drive relapse therapy discontinuation chronic myeloid leukemia (CML) built map, detailing - from receptor key phenotypes molecular implicated control proliferation, DNA damage response inflammation In-depth analysis map uncovered vulnerabilities. Functional validation patient-derived leukemic stem revealed acquired FLT3-dependency its underlying mechanism. In conclusion, our study presents generally applicable reposition FLT3, one most frequently mutated drivers acute leukemia, as potential target CML

Язык: Английский

Процитировано

0
High-Throughput Workflow for Detergent-free Cell-Based Proteomic Characterization DOI
Saeed Seyedmohammad, Alejandro Rivas,

Maxim Zhgamadze

и другие.

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

Опубликована: Апрель 21, 2025

We have developed an automated cell-based workflow for the quantification of proteins by liquid chromatography-mass spectrometry (LC-MS) that facilitates large-scale perturbation studies carried out in a 96-well plate format and enables preparation one full approximately 4 h, showcasing high-throughput (HTP) concept. Cells were grown lysed via ultrasonication. Proteins subsequently solubilized, extracted, processed into tryptic peptides 2 h before being acquired data-independent acquisition mass (DIA-MS). This leverages adaptive focused acoustics (AFA) technology ultrasonication to aid cell lysis protein solubilization on handling platform. As proof principle, AC16 human cardiomyocyte-like cells cultured under optimized conditions compatible with downstream HTP pipeline. Over 30,000 identified, corresponding detection 5100 unique proteins. 50% measured had average coefficient variation (CV) 25% from cells. Our detergent-free buffer consisting ammonium bicarbonate yielded comparable findings. For same number cells, 5000 identified 29,000 peptides, 40% which demonstrated CV 25%.

Язык: Английский

Процитировано

0
Understanding the molecular diversity of synapses DOI
Marc van Oostrum, Beatriz Álvarez-Castelao

Nature reviews. Neuroscience, Год журнала: 2024, Номер unknown

Опубликована: Дек. 5, 2024

Язык: Английский

Процитировано

2
Phosphoproteomics highlights complex resource management upon inflammatory stimulation of fibroblasts DOI Creative Commons
Patricia Bortel, Ana Martínez‐Val, Gerhard Hagn

и другие.

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

Опубликована: Окт. 11, 2024

ABSTRACT The stimulation of cells by inflammatory mediators gives rise to intricate signaling cascades, inducing specific biological functions. role kinase activation for the establishment functions has only scarcely been established. A time-course analysis human fetal fibroblasts stimulated with Interleukin-1β (IL-1β) and/or Dexamethasone (Dex) was conducted using mass spectrometry-based proteomics and phosphoproteomics in conjunction lysolipid oxylipin profiling. IL-1β induced proteome alterations indicated metabolic, transcriptional, translational activation, including marker proteins such as NOS1, THBS1, STING1. induction mitochondrial formation numerous lysolipids an increase beta-oxidation. In addition NF-κB STAT pathways, which are characteristic MAP AKT pathways were found be strongly induced. Six hours after treatment, observed events exhibited a notable decline, nearly returning their initial states 24 hours. It is noteworthy that all these activities also treated Dex alone. Additionally, transient alterations, included otherwise response, MMP3 NFKB2. Activation PIKFYVE apparently dexamethasone but constituted minority total phosphorylation events. Only glucocorticoids, TSC22D3 MAOA, observed. effects on background established verified known inhibitory resulted expression anti-inflammatory oxylipins, hardly affected involving kinases. conclusion, this data demonstrates majority inflammation-associated needs attributed resource stress management rather than effector

Язык: Английский

Процитировано

0
EasyAb: A High-Throughput Workflow for Antibody-Based PTM Peptide Enrichment Method Coupled to Mass Spectrometry DOI Creative Commons
Ashok Kumar Jayavelu, Jeffrey J. Liu, Sonja C. Schriever

и другие.

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

Опубликована: Ноя. 1, 2024

SUMMARY Reversible post-translational modification (PTMs) is a fundamental mechanism of cellular signal transduction. In vivo and ex studies to profile PTMs have greatly advanced our understanding the complexities signaling. However, apart from most commonly studied PTMs, large-scale analysis still very challenging, limiting various processes. PTM-bearing peptides often enriched by antibodies, followed unbiased mass spectrometry (MS)-based readout hundreds or thousands sites. To extend reach this powerful technology in with small protein starting amounts, we here developed EasyAb, streamlined high throughput MS workflow for antibody-based PTM profiling. Using epidermal growth factor receptor (EGFR) signaling acute myeloid leukemia (AML) cell systems, demonstrate that EasyAb increases sensitivity enables multiple systems-level studies. Furthermore, resolves brain G protein-coupled receptor-mediated tyrosine kinase activation reveals long elusive hypothalamic neuron-specific leptin architecture. Graphical Abstract HIGHLIGHTS sensitive, rapid high-throughput method quantify post translationally modified diverse systems. differential phosphorylation mRNA splicing proteins AML patient samples. Activation KOR “G-protein-biased” non-aversive agonist 6’GNTI elicits Src activity mice. Elucidation Leptin induced LEPRb-Jak2 phosphotyrosine

Язык: Английский

Процитировано

0
Proteogenomic profiling of acute myeloid leukemia to identify therapeutic targets DOI
Heather C. Murray, Jonathan R. Sillar, Melissa G. Chambers

и другие.

Expert Review of Proteomics, Год журнала: 2024, Номер unknown, С. 1 - 14

Опубликована: Ноя. 22, 2024

Acute myeloid leukemia (AML) is an aggressive and poor-prognosis blood cancer. Despite a low mutation burden compared to other cancers, AML heterogenous identifying robust therapeutic targets has been difficult. Genomic profiling greatly advanced our understanding of AML, revealed for therapy. However, only 50% patients have gene mutations that are currently druggable, relapse rates remain high. The addition proteomic emerging address these challenges.

Язык: Английский

Процитировано

0
Advancements in Global Phosphoproteomics Profiling: Overcoming Challenges in Sensitivity and Quantification DOI Creative Commons
Gul Muneer,

Ciao‐Syuan Chen,

Yu‐Ju Chen

и другие.

PROTEOMICS, Год журнала: 2024, Номер unknown

Опубликована: Дек. 18, 2024

Protein phosphorylation introduces post-genomic diversity to proteins, which plays a crucial role in various cellular activities. Elucidation of system-wide signaling cascades requires high-performance tools for precise identification and quantification dynamics site-specific events. Recent advances phosphoproteomic technologies have enabled the comprehensive mapping dynamic landscape, has opened new avenues exploring cell type-specific functional networks underlying functions clinical phenotypes. Here, we provide an overview basics challenges phosphoproteomics, as well technological evolution current state-of-the-art global quantitative phosphoproteomics methodologies. With specific focus on highly sensitive platforms, summarize recent trends innovations miniaturized sample preparation strategies micro-to-nanoscale single-cell profiling, data-independent acquisition mass spectrometry (DIA-MS) enhanced coverage, pipelines deep disease biology. Each aspect analysis presents unique opportunities improvement innovation. We specifically highlight evolving that enable profiling from low-input samples. Finally, discuss persistent technologies, including feasibility nanoscale future outlooks biomedical applications.

Язык: Английский

Процитировано

0