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1.
bioRxiv ; 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38645098

RESUMO

A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow from planning to analysis. We share real-world case studies applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at protein and peptide-level allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis using Skyline, longitudinal QC metrics using AutoQC, and server-based data deposition using PanoramaWeb. We propose that this integrated approach to QC be used as a starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible.

2.
bioRxiv ; 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38617345

RESUMO

Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 µL plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimer's disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinson's disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 ± 0.06.

3.
Elife ; 122023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36799304

RESUMO

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue- and mutation type-dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.


Assuntos
Envelhecimento , DNA Mitocondrial , Camundongos , Animais , DNA Mitocondrial/genética , Envelhecimento/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/genética , Mutação
4.
Invest Ophthalmol Vis Sci ; 62(14): 20, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34797906

RESUMO

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.


Assuntos
Envelhecimento/fisiologia , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Visão de Cores/fisiologia , Eletrorretinografia , Fusão Flicker/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Glutamina/metabolismo , Luz , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Visão Noturna/fisiologia , Consumo de Oxigênio/fisiologia , Estimulação Luminosa
5.
Nucleic Acids Res ; 49(19): 11103-11118, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34614167

RESUMO

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.


Assuntos
Envelhecimento/genética , Replicação do DNA , DNA Mitocondrial/genética , Genoma Mitocondrial , Mutação em Linhagem Germinativa , Mitocôndrias/genética , Acúmulo de Mutações , Envelhecimento/metabolismo , Animais , Mapeamento Cromossômico , DNA Polimerase gama/deficiência , DNA Polimerase gama/genética , DNA Mitocondrial/metabolismo , Especiação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Taxa de Mutação , Polimorfismo de Nucleotídeo Único
6.
Breast Cancer Res ; 22(1): 135, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267867

RESUMO

BACKGROUND: The lack of specificity and high degree of false positive and false negative rates when using mammographic screening for detecting early-stage breast cancer is a critical issue. Blood-based molecular assays that could be used in adjunct with mammography for increased specificity and sensitivity could have profound clinical impact. Our objective was to discover and independently verify a panel of candidate blood-based biomarkers that could identify the earliest stages of breast cancer and complement current mammographic screening approaches. METHODS: We used affinity hydrogel nanoparticles coupled with LC-MS/MS analysis to enrich and analyze low-abundance proteins in serum samples from 20 patients with invasive ductal carcinoma (IDC) breast cancer and 20 female control individuals with positive mammograms and benign pathology at biopsy. We compared these results to those obtained from five cohorts of individuals diagnosed with cancer in organs other than breast (ovarian, lung, prostate, and colon cancer, as well as melanoma) to establish IDC-specific protein signatures. Twenty-four IDC candidate biomarkers were then verified by multiple reaction monitoring (LC-MRM) in an independent validation cohort of 60 serum samples specifically including earliest-stage breast cancer and benign controls (19 early-stage (T1a) IDC and 41 controls). RESULTS: In our discovery set, 56 proteins were increased in the serum samples from IDC patients, and 32 of these proteins were specific to IDC. Verification of a subset of these proteins in an independent cohort of early-stage T1a breast cancer yielded a panel of 4 proteins, ITGA2B (integrin subunit alpha IIb), FLNA (Filamin A), RAP1A (Ras-associated protein-1A), and TLN-1 (Talin-1), which classified breast cancer patients with 100% sensitivity and 85% specificity (AUC of 0.93). CONCLUSIONS: Using a nanoparticle-based protein enrichment technology, we identified and verified a highly specific and sensitive protein signature indicative of early-stage breast cancer with no false positives when assessing benign and inflammatory controls. These markers have been previously reported in cell-ECM interaction and tumor microenvironment biology. Further studies with larger cohorts are needed to evaluate whether this biomarker panel improves the positive predictive value of mammography for breast cancer detection.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Detecção Precoce de Câncer/métodos , Proteínas da Matriz Extracelular/sangue , Adulto , Idoso , Biópsia , Mama/diagnóstico por imagem , Mama/patologia , Neoplasias da Mama/sangue , Carcinoma Ductal de Mama/sangue , Carcinoma Ductal de Mama/patologia , Estudos de Casos e Controles , Estudos de Coortes , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Masculino , Mamografia , Pessoa de Meia-Idade , Nanopartículas/química , Proteômica/métodos
7.
Proc Natl Acad Sci U S A ; 117(46): 28816-28827, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33144507

RESUMO

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Ritmo Circadiano/fisiologia , Adaptação à Escuridão/fisiologia , Retículo Endoplasmático/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Peixe-Zebra
8.
Cell Death Dis ; 9(2): 240, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29445082

RESUMO

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that is expressed in cancer cells. Its role in tumor metabolism is not definitively established, but investigators have suggested that regulation of PKM2 activity can cause accumulation of glycolytic intermediates and increase flux through the pentose phosphate pathway. Recent evidence suggests that PKM2 also may have non-metabolic functions, including as a transcriptional co-activator in gene regulation. We reported previously that PKM2 is abundant in photoreceptor cells in mouse retinas. In the present study, we conditionally deleted PKM2 (rod-cre PKM2-KO) in rod photoreceptors and found that the absence of PKM2 causes increased expression of PKM1 in rods. Analysis of metabolic flux from U-13C glucose shows that rod-cre PKM2-KO retinas accumulate glycolytic intermediates, consistent with an overall reduction in the amount of pyruvate kinase activity. Rod-cre PKM2-KO mice also have an increased NADPH availability could favor lipid synthesis, but we found no difference in phospholipid synthesis between rod-cre PKM2 KO and PKM2-positive controls. As rod-cre PKM2-KO mice aged, we observed a significant loss of rod function, reduced thickness of the photoreceptor outer segment layer, and reduced expression of photoreceptor proteins, including PDE6ß. The rod-cre PKM2-KO retinas showed greater TUNEL staining than wild-type retinas, indicating a slow retinal degeneration. In vitro analysis showed that PKM2 can regulate transcriptional activity from the PDE6ß promoter in vitro. Our findings indicate that both the metabolic and transcriptional regulatory functions of PKM2 may contribute to photoreceptor structure, function, and viability.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Piruvato Quinase/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Transcrição Gênica , Animais , Apoptose/genética , Isótopos de Carbono , Membrana Celular/química , Membrana Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , NADP/metabolismo , Fosfolipídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/deficiência , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Transdução de Sinais , Coloração e Rotulagem/métodos , Tomografia de Coerência Óptica , Triglicerídeos/metabolismo
9.
Elife ; 62017 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-28901286

RESUMO

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.


Assuntos
Adaptação Ocular , Metabolismo Energético , Células Ependimogliais/fisiologia , Células Fotorreceptoras/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Células Ependimogliais/metabolismo , Glucose/metabolismo , Humanos , Lactatos/metabolismo , Camundongos , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Peixe-Zebra
10.
Brain Res ; 1647: 79-93, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-26979993

RESUMO

The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized the protein-protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/FTLD. Using immunoprecipitation coupled to mass spectrometry (IP-MS), we identified 132 proteins that specifically interact with RBM45 within HEK293 cells. Select PPIs were validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. Analysis of the biological processes and pathways associated with RBM45-interacting proteins indicates enrichment for nuclear RNA processing/splicing via association with hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact with RBM45, consistent with prior observations of these proteins within intracellular inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, suggest novel functions for this protein, and provide new insights into the contributions of RBM45 to neurodegeneration in ALS/FTLD. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Corpos de Inclusão/metabolismo , Espectrometria de Massas , Fatores de Iniciação de Peptídeos/metabolismo , Mapas de Interação de Proteínas , Splicing de RNA , Transdução de Sinais
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