Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

ACS Measurement Science Au, Год журнала: 2024, Номер 4(4), С. 338 - 417

Опубликована: Июнь 4, 2024

Proteomics is the large scale study of protein structure and function from biological systems through identification quantification."Shotgun proteomics" or "bottom-up prevailing strategy, in which proteins are hydrolyzed into peptides that analyzed by mass spectrometry.Proteomics studies can be applied to diverse ranging simple proteoforms, protein-protein interactions, structural alterations, absolute relative quantification, post-translational modifications, stability.To enable this range different experiments, there strategies for proteome analysis.The nuances how proteomic workflows differ may challenging understand new practitioners.Here, we provide a comprehensive overview proteomics methods.We cover biochemistry basics extraction interpretation orthogonal validation.We expect Review will serve as handbook researchers who field bottom-up proteomics.

Язык: Английский

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Proteomics of the heart

Physiological Reviews, Год журнала: 2024, Номер 104(3), С. 931 - 982

Опубликована: Фев. 1, 2024

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at molecular level. Proteomics provides biologists with snapshot of context-dependent and proteoform expression, structural conformations, dynamic turnover, protein-protein interactions. Cardiac can offer broader deeper understanding the mechanisms that underscore cardiovascular disease, it foundational to development future therapeutic interventions. This review encapsulates evolution, current technologies, perspectives proteomic-based mass spectrometry as applies study heart. Key technological advancements have allowed researchers proteomes single-cell level employ robot-assisted automation systems for enhanced sample preparation techniques, increase fidelity spectrometers has unambiguous numerous posttranslational modifications. Animal models ranging from early animal experiments heart failure preserved ejection fraction, provided tools challenging organ laboratory. Further will pave way implementation even closer within clinical setting, allowing not only scientists but also patients benefit an interplay relates cardiac disease physiology.

Язык: Английский

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Proteomic Profiling Towards a Better Understanding of Genetic Based Muscular Diseases: The Current Picture and a Look to the Future

Biomolecules, Год журнала: 2025, Номер 15(1), С. 130 - 130

Опубликована: Янв. 15, 2025

Proteomics accelerates diagnosis and research of muscular diseases by enabling the robust analysis proteins relevant for manifestation neuromuscular in following aspects: (i) evaluation effect genetic variants on corresponding protein, (ii) prediction underlying defect based proteomic signature muscle biopsies, (iii) pathophysiologies different entities diseases, key definition new intervention concepts, (iv) patient stratification according to biochemical fingerprints as well (v) monitoring success therapeutic interventions. This review presents—also through exemplary case studies—the various advantages mass proteomics investigation discusses technical limitations, provides an outlook possible future application concepts. Hence, is excellent large-scale analytical tool diagnostic workup (hereditary) warrants systematic profiling pathophysiological processes. The steady development may allow overcome existing limitations including a quenched dynamic range quantification protein isoforms. Future directions include targeted settings using not only biopsies but also liquid address need minimally invasive procedures.

Язык: Английский

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Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25

Journal of Proteome Research, Год журнала: 2024, Номер 23(4), С. 1285 - 1297

Опубликована: Март 13, 2024

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise humans. Phosphorylation improve ex vivo muscle contractile function mice, but our understanding of molecular mechanisms is incomplete. Here, we profiled interactome mouse myotubes using affinity purification coupled mass spectrometry. This analysis included an investigation AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic contractile-associated proteins were identified, which revealed a subset GTPases that associate with AMPK- phosphorylation-dependent manner. We confirmed localized nucleus apparatus muscle. Mice lacking

Язык: Английский

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Cell Storage Conditions Impact Single-Cell Proteomic Landscapes

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

Опубликована: Янв. 24, 2025

Single cell transcriptomics (SCT) has revolutionized our understanding of cellular heterogeneity, yet the emergence single proteomics (SCP) promises a more functional view dynamics. A challenge is that not all mass spectrometry facilities can perform SCP, and laboratories have access to sorting equipment required for which together motivate an interest in sending bulk samples through mail SCP analysis. Shipping requires storage, unknown effect on results. This study investigates impact storage conditions proteomic landscape at level, utilizing Data-Independent Acquisition (DIA) coupled with Parallel Accumulation Serial Fragmentation (diaPASEF). Three were compared 293T cells: (1) 37 °C (control), (2) 4 overnight, (3) −196 followed by liquid nitrogen preservation. Both cold frozen induced significant alterations diameter, elongation, proteome composition. By elucidating how alter morphology profiles, this contributes foundational technical information about sample preparation data quality.

Язык: Английский

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Human skeletal muscle fiber heterogeneity beyond myosin heavy chains

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Фев. 19, 2025

Skeletal muscle is a heterogenous tissue comprised primarily of myofibers, commonly classified into three fiber types in humans: one "slow" (type 1) and two "fast" 2A type 2X). However, heterogeneity between within traditional remains underexplored. We applied transcriptomic proteomic workflows to 1050 1038 single myofibers from human vastus lateralis, respectively. Proteomics was conducted males, while transcriptomics included ten males females. identify metabolic, ribosomal, cell junction proteins, addition myosin heavy chain isoforms, as sources multi-dimensional variation myofibers. Furthermore, whilst slow fast clusters are identified, our data suggests that 2X fibers not phenotypically distinct other fibers. Moreover, chain-based classifications do adequately describe the phenotype nemaline myopathy. Overall, indicates myofiber with beyond isoforms. Moreno-Justicia colleagues report pipelines explore skeletal fibers, identifying healthy individuals children

Язык: Английский

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Fiber Type–Specific Adaptations to Exercise Training in Human Skeletal Muscle: Lessons From Proteome Analyses and Future Directions

Scandinavian Journal of Medicine and Science in Sports, Год журнала: 2025, Номер 35(5)

Опубликована: Апрель 25, 2025

ABSTRACT Skeletal muscle is a key determinant of sports performance. It highly specialized, yet complex and heterogeneous tissue, comprising multiple cell types. Muscle fibers are the main functional type responsible for converting energy into mechanical work. They exhibit remarkable ability to adapt in response stressors, such as exercise training. But while it recognized that human skeletal have distinct contractile metabolic features, classified slow/oxidative (type 1) or fast/glycolytic 2a/x), less attention has been directed adaptability different fiber Methodological advancements mass spectrometry‐based proteomics allow researchers quantify thousands proteins with only small amount tissue—even single fiber. By exploiting this technology, studies emerging highlighting subpopulations differently This review provides contemporary perspective on type–specific training humans. A aim our facilitate further within physiology by harnessing spectrometry proteomics.

Язык: Английский

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How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

Proteomes, Год журнала: 2024, Номер 12(1), С. 4 - 4

Опубликована: Янв. 16, 2024

This perspective article is concerned with the question of how proteomics, which a core technique systems biology that deeply embedded in multi-omics field modern bioresearch, can help us better understand molecular pathogenesis complex diseases. As an illustrative example monogenetic disorder primarily affects neuromuscular system but characterized by plethora multi-system pathophysiological alterations, muscle-wasting disease Duchenne muscular dystrophy was examined. Recent achievements dystrophinopathy research are described special reference to proteome-wide complexity changes and body-wide alterations/adaptations. Based on description current applications top-down versus bottom-up proteomic approaches their technical challenges, future biological outlined. The envisaged holistic integromic bioanalysis would encompass integration diverse omics-type studies including inter- intra-proteomics as disciplines for systematic protein evaluations, sophisticated biomolecular analyses, physiology, biology, biochemistry histochemistry. Integrated findings promise be instrumental improving our detailed knowledge pathogenic mechanisms dysfunction, widening available biomarker signature improved diagnostic/prognostic procedures, advancing identification novel therapeutic targets treat dystrophy.

Язык: Английский

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Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

Cells, Год журнала: 2023, Номер 12(21), С. 2560 - 2560

Опубликована: Ноя. 1, 2023

Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle has been extensively studied in relation myogenesis, fiber type specification, transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, co-morbidities sarcopenia old age. Since tissue accounts for approximately 40% body mass humans, alterations have considerable influence on whole-body physiology. This review outlines main bioanalytical avenues taken proteomic characterization tissues, including top-down proteomics focusing intact proteoforms their post-translational modifications, bottom-up proteomics, which is peptide-centric method concerned with large-scale detection proteins mixtures, subproteomics examines protein composition distinct subcellular fractions. Mass spectrometric studies over last two decades decisively improved our general cell biological understanding diversity heterogeneous individual myofibers muscles. detailed knowledge can now be integrated findings from other omics-type methodologies establish systems view function.

Язык: Английский

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The current economics and throughput of single cell proteomics by liquid chromatography mass spectrometry

Опубликована: Июнь 11, 2024

Single cell proteomics by mass spectrometry (SCP) is an emerging field of study that has captured the interest and imagination biologists in a wide array disciplines. In pursuit this new dizzying technologies techniques have demonstrated ability to quantify hundreds few thousand proteins single mammalian cells typical size. One striking characteristic these methods range relative costs associated with analysis each cell. We attempted estimate cost per across 17 different studies based on quotes we obtained for hardware, reagents instrument support plans relation number can be analyzed day. Before including labor or facilities, find analyze size from less than <$2 over $50 The increase appears directly related decrease throughput as measured theoretical maximum Perhaps most surprising observation average year. This when compared emergence RNA sequencing where increased, cost/cell decreased exponentially first 7 years field’s emergence. While made many assumptions obtain estimates, hope will informative scientists interested obtaining SCP data spectrometrists who are considering entering field. provided spreadsheet simple calculator supplemental allow others adjust our calculations other variables which inevitably described future.

Язык: Английский

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