RESUMEN
Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to â¼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify â¼1000 proteins from no more than 1 µg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.
Asunto(s)
Proteoma , Proteómica , Humanos , Proteoma/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Programas InformáticosRESUMEN
Identification and proteomic characterization of rare cell types within complex organ-derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 min of total run time per cell with peptides quantified over 11.5 min using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single cells per day. At this throughput, nanoDTSC quantified over 1000 proteins in individual cardiomyocytes and heterogeneous populations of single cells from the aorta.
Asunto(s)
Proteínas , Proteómica , Proteómica/métodos , Cromatografía Liquida/métodos , Proteínas/química , Péptidos/química , Espectrometría de Masas/métodosRESUMEN
Single-cell omics data analysis pipelines are complicated to design and difficult to share or reproduce. We describe a web platform that enables no-code analysis pipeline design, simple computing via the Open Science Grid, and sharing of entire data analysis pipelines, their input data, and interactive results. We expect this platform to increase the accessibility and reproducibility of single-cell omics.
RESUMEN
Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.
Asunto(s)
Proteoma , Proteómica , Humanos , Proteoma/metabolismo , Proteómica/métodos , Flujo de Trabajo , Fibras Musculares Esqueléticas/metabolismo , Músculo EsqueléticoRESUMEN
Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.
RESUMEN
Identification and proteomic characterization of rare cell types within complex organ derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 minutes of total run time per cell with peptides quantified over 11.5 minutes using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single-cells per day. At this throughput, nanoDTSC quantified over 1,000 proteins in individual cardiomyocytes and heterogenous populations of single cells from aorta.
RESUMEN
BACKGROUND AND OBJECTIVES: Functional outcomes after stroke are strongly related to focal injury measures. However, the role of global brain health is less clear. In this study, we examined the impact of brain age, a measure of neurobiological aging derived from whole-brain structural neuroimaging, on poststroke outcomes, with a focus on sensorimotor performance. We hypothesized that more lesion damage would result in older brain age, which would in turn be associated with poorer outcomes. Related, we expected that brain age would mediate the relationship between lesion damage and outcomes. Finally, we hypothesized that structural brain resilience, which we define in the context of stroke as younger brain age given matched lesion damage, would differentiate people with good vs poor outcomes. METHODS: We conducted a cross-sectional observational study using a multisite dataset of 3-dimensional brain structural MRIs and clinical measures from the ENIGMA Stroke Recovery. Brain age was calculated from 77 neuroanatomical features using a ridge regression model trained and validated on 4,314 healthy controls. We performed a 3-step mediation analysis with robust mixed-effects linear regression models to examine relationships between brain age, lesion damage, and stroke outcomes. We used propensity score matching and logistic regression to examine whether brain resilience predicts good vs poor outcomes in patients with matched lesion damage. RESULTS: We examined 963 patients across 38 cohorts. Greater lesion damage was associated with older brain age (ß = 0.21; 95% CI 0.04-0.38, p = 0.015), which in turn was associated with poorer outcomes, both in the sensorimotor domain (ß = -0.28; 95% CI -0.41 to -0.15, p < 0.001) and across multiple domains of function (ß = -0.14; 95% CI -0.22 to -0.06, p < 0.001). Brain age mediated 15% of the impact of lesion damage on sensorimotor performance (95% CI 3%-58%, p = 0.01). Greater brain resilience explained why people have better outcomes, given matched lesion damage (odds ratio 1.04, 95% CI 1.01-1.08, p = 0.004). DISCUSSION: We provide evidence that younger brain age is associated with superior poststroke outcomes and modifies the impact of focal damage. The inclusion of imaging-based assessments of brain age and brain resilience may improve the prediction of poststroke outcomes compared with focal injury measures alone, opening new possibilities for potential therapeutic targets.
Asunto(s)
Accidente Cerebrovascular , Humanos , Anciano , Estudios Transversales , Accidente Cerebrovascular/complicaciones , Encéfalo/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , NeuroimagenRESUMEN
Magnetic resonance imaging (MRI) is an important imaging modality in stroke. Computer based automated medical image processing is increasingly finding its way into clinical routine. The Ischemic Stroke Lesion Segmentation (ISLES) challenge is a continuous effort to develop and identify benchmark methods for acute and sub-acute ischemic stroke lesion segmentation. Here we introduce an expert-annotated, multicenter MRI dataset for segmentation of acute to subacute stroke lesions ( https://doi.org/10.5281/zenodo.7153326 ). This dataset comprises 400 multi-vendor MRI cases with high variability in stroke lesion size, quantity and location. It is split into a training dataset of n = 250 and a test dataset of n = 150. All training data is publicly available. The test dataset will be used for model validation only and will not be released to the public. This dataset serves as the foundation of the ISLES 2022 challenge ( https://www.isles-challenge.org/ ) with the goal of finding algorithmic methods to enable the development and benchmarking of automatic, robust and accurate segmentation methods for ischemic stroke.
Asunto(s)
Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Procesamiento de Imagen Asistido por Computador/métodos , BenchmarkingRESUMEN
Accurate lesion segmentation is critical in stroke rehabilitation research for the quantification of lesion burden and accurate image processing. Current automated lesion segmentation methods for T1-weighted (T1w) MRIs, commonly used in stroke research, lack accuracy and reliability. Manual segmentation remains the gold standard, but it is time-consuming, subjective, and requires neuroanatomical expertise. We previously released an open-source dataset of stroke T1w MRIs and manually-segmented lesion masks (ATLAS v1.2, N = 304) to encourage the development of better algorithms. However, many methods developed with ATLAS v1.2 report low accuracy, are not publicly accessible or are improperly validated, limiting their utility to the field. Here we present ATLAS v2.0 (N = 1271), a larger dataset of T1w MRIs and manually segmented lesion masks that includes training (n = 655), test (hidden masks, n = 300), and generalizability (hidden MRIs and masks, n = 316) datasets. Algorithm development using this larger sample should lead to more robust solutions; the hidden datasets allow for unbiased performance evaluation via segmentation challenges. We anticipate that ATLAS v2.0 will lead to improved algorithms, facilitating large-scale stroke research.