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

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(10), P. 2374 - 2380

Published: Aug. 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

Language: Английский

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Protein aggregation and therapeutic strategies in SOD1- and TDP-43- linked ALS

Frontiers in Molecular Biosciences, Journal Year: 2024, Volume and Issue: 11

Published: May 24, 2024

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with severe socio-economic impact. A hallmark of ALS pathology the presence aberrant cytoplasmic inclusions composed misfolded and aggregated proteins, including both wild-type mutant forms. This review highlights critical role protein species in pathogenesis, particularly focusing on Cu/Zn superoxide dismutase (SOD1) TAR DNA-binding 43 (TDP-43), emphasizes urgent need for innovative therapeutic strategies targeting these proteins directly. Despite significant advancements understanding mechanisms, remains incurable, current treatments offering limited clinical benefits. Through comprehensive analysis, focuses direct modulation presents recent discoveries small molecules peptides that inhibit SOD1 TDP-43 aggregation, underscoring their potential as effective to modify progression improve outcomes.

Language: Английский

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Annexin A11 aggregation in FTLD–TDP type C and related neurodegenerative disease proteinopathies

Acta Neuropathologica, Journal Year: 2024, Volume and Issue: 147(1)

Published: June 1, 2024

Abstract TAR DNA-binding protein 43 (TDP-43) is an RNA binding found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with (FTLD–TDP) and limbic predominant age-related encephalopathy neuropathologic change (LATE-NC). Annexin A11 also known form aggregates ALS cases pathogenic variants ANXA11 . aggregation has not been described sporadic ALS, FTLD–TDP or LATE-NC cases. To explore the relationship between A11, genetic analysis of 822 autopsy was performed identify rare variants. In addition, immunohistochemical study 368 aggregates. Insoluble which colocalize were present all Type C seen a small proportion (3–6%) types A B, LATE-NC. we confirm comingling case p.G38R variant. Finally, abundant as primary pathologic finding progressive supranuclear palsy-like dementia prominent striatal vacuolization due novel variant, p.P75S. By immunoblot, annexinopathy variant show accumulation insoluble truncated fragment. These results indicate that diverse heterogeneous range both proteinopathies. vacuolar p.P75S suggests sufficient cause neurodegeneration.

Language: Английский

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Exosomes in Regulating miRNAs for Biomarkers of Neurodegenerative Disorders

Molecular Neurobiology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 7, 2025

Language: Английский

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

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(26), P. 10534 - 10542

Published: June 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.

Language: Английский

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Regulation of cortical hyperexcitability in amyotrophic lateral sclerosis: focusing on glial mechanisms

Molecular Neurodegeneration, Journal Year: 2023, Volume and Issue: 18(1)

Published: Oct. 19, 2023

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of both upper and lower motor neurons, resulting in muscle weakness, atrophy, paralysis, eventually death. Motor cortical hyperexcitability common phenomenon observed at presymptomatic stage ALS. Both cell-autonomous (the intrinsic properties neurons) non-cell-autonomous mechanisms (cells other than are believed to contribute hyperexcitability. Decoding pathological relevance these dynamic changes neurons glial cells has remained major challenge. This review summarizes evidence from clinical preclinical research, as well underlying mechanisms. We discuss potential role cells, particularly microglia, regulating abnormal neuronal activity during disease progression. Identifying early such system may provide new insights for earlier diagnosis ALS reveal novel targets halt

Language: Английский

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Biomarkers for Managing Neurodegenerative Diseases

Biomolecules, Journal Year: 2024, Volume and Issue: 14(4), P. 398 - 398

Published: March 26, 2024

Neurological disorders are the leading cause of cognitive and physical disability worldwide, affecting 15% global population. Due to demographics aging, prevalence neurological disorders, including neurodegenerative diseases, will double over next two decades. Unfortunately, while available therapies provide symptomatic relief for motor impairment, there is an urgent unmet need develop disease-modifying that slow rate pathological progression. In context, biomarkers could identify at-risk prodromal patients, monitor disease progression, track responses therapy, parse causality molecular events novel targets further clinical investigation. Thus, identifying discriminate between diseases reflect specific stages pathology would catalyze discovery development therapeutic targets. This review describe prevalence, known mechanisms, ongoing or recently concluded trials, three most prevalent Alzheimer’s (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s (PD).

Language: Английский

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Omics studies of nuclear protein aggregates in subcellular fractions reveals co- aggregation of RNA-binding proteins affecting cytosolic pathways

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 13, 2025

Disease-associated RNA binding protein (RBP) aggregation is a hallmark of several age-related neurodegenerative diseases. How insoluble RBP aggregates leads to cellular dysfunction poorly understood. Here, we investigated the molecular mechanisms affected by PABPN1 aggregates. are nuclear, but regulates nuclear export mRNA. To explore consequences aggregates, performed sequencing and proteomic studies in subcellular fractions an inducible human muscle cell model. analyses revealed this model associated with reduced endogenous levels. Proteomic that most changes driven were cytoplasmic fraction, accounting for metabolism, differentiation biomechanics. Changes fraction small enriched RBPs. We show sequestration mRNA impaired translational efficiency. Our study suggests RBPs regulated both gain-of-function loss-of-function mechanisms, which relevant development therapeutics age-associated

Language: Английский

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Unlocking Disease‐Modifying Treatments for TDP‐43‐Mediated Neurodegeneration

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

Published: Feb. 3, 2025

Neurons degenerate in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), causing progressive inevitably fatal neurological decline. The best therapeutic strategies target underlying disease mediators, but after decades of intensive research, the causes these neurodegenerative diseases remain elusive. Recently, coordinated activities large consortia, increasing open access to datasets, new methods such as cryo-transmission electron microscopy, advancements high-resolution omics technologies have offered insights into biology that bring us closer understanding mechanisms neurodegeneration. In particular, improved roles key pathological protein TAR DNA binding 43 (TDP-43) has revealed intriguing opportunities provide hope for better diagnostic tools effective treatments ALS FTD.

Language: Английский

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Mitochondrial respiratory complex IV deficiency recapitulates amyotrophic lateral sclerosis

Nature Neuroscience, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

Language: Английский

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