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Proteomics Oct 2022Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine... (Review)
Review
Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.
Topics: Proteomics; Proteome; Tandem Mass Spectrometry; Isotope Labeling; Peptides; Biomarkers; Lipids
PubMed: 35687565
DOI: 10.1002/pmic.202100256 -
International Journal of Gynecological... Dec 2009To describe the role of proteomic profiling in the diagnosis and treatment of ovarian cancer. (Review)
Review
OBJECTIVE
To describe the role of proteomic profiling in the diagnosis and treatment of ovarian cancer.
METHODS
We report a thorough review of the literature, ongoing trials, and our group's experience with proteomic profiling for early detection, recurrence, and treatment of ovarian cancer.
RESULTS/CONCLUSIONS
Ovarian cancer remains the deadliest gynecologic malignancy in the western world and is most often diagnosed at a rarely curable late stage. Novel applications of proteomic techniques, such as mass spectrometry, show promise in the quest for reliable multimodality screening programs for the early detection of ovarian cancer. Proteomic analysis of tissue samples has underscored the heterogeneity of this disease process. Development of validated assays that survey the genetic and/or proteomic makeup of an individual tumor will add greatly to the histological classification of the tumor and may lead to different treatment approaches tailored to the unique expression pattern of each individual patient. As novel agents that disrupt signal propagation develop, proteomic profiling by reverse-phase protein arrays can characterize the in-tumor efficacy of the agent by quantification of the changes in expression levels of activated proteins. Together, better understanding of the potential diagnostic and therapeutic targets followed with proof-of-target effect will lead to rational combinations of novel therapy and improve individual ovarian cancer patient outcome.
Topics: Biomarkers, Tumor; Carcinoma; Early Detection of Cancer; Female; Humans; Models, Biological; Ovarian Neoplasms; Proteome; Proteomics
PubMed: 19955909
DOI: 10.1111/IGC.0b013e3181c03929 -
Brazilian Journal of Biology = Revista... Feb 2018Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In...
Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.
Topics: Electrophoresis, Gel, Two-Dimensional; Piper; Plant Proteins; Proteome; Proteomics; Spectrometry, Mass, Electrospray Ionization
PubMed: 28699966
DOI: 10.1590/1519-6984.07816 -
Investigative Ophthalmology & Visual... Oct 2023The purpose of this study was to investigate the underlying molecular mechanism of lens-induced myopia (LIM) through transcriptome and proteome analyses with a modified...
PURPOSE
The purpose of this study was to investigate the underlying molecular mechanism of lens-induced myopia (LIM) through transcriptome and proteome analyses with a modified mouse myopia model.
METHODS
Four-week-old C57BL/6J mice were treated with a homemade newly designed -25 diopter (D) lens mounting by a 3D printing pen before right eyes for 4 weeks. Refraction (RE) and axial dimensions were measured every 2 weeks. Retinas were analyzed by RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, and STRING databases were used to identify significantly affected pathways in transcriptomic and proteomic data sets. Western blot was used to detect the expression of specific proteins.
RESULTS
The modified model was accessible and efficient. Mice displayed a significant myopic shift (approximately 8 D) following 4 weeks' of lens treatment. Through transcriptomics and proteomics analysis, we elucidated 175 differently expressed genes (DEGs) and 646 differentially expressed proteins (DEPs) between binoculus. The transcriptomic and proteomic data showed a low correlation. Going over the mRNA protein matches, insulin like growth factor 2 mRNA binding protein 1 (Igf2bp1) was found to be a convincing biomarker of LIM, which was confirmed by Western blot. RNA-seq and proteome profiling confirmed that these two "omics" data sets complemented one another in KEGG pathways annovation. Among these, metabolic and human diseases pathways were considered to be correlated with the LIM forming process.
CONCLUSIONS
The newly constructed LIM model provides a useful tool for future myopia research. Combining transcriptomic and proteomic analysis may potentially brighten the prospects of novel therapeutic targets for patients with myopia.
Topics: Animals; Humans; Mice; Transcriptome; Proteome; Proteomics; Mice, Inbred C57BL; Disease Models, Animal; RNA, Messenger; Myopia
PubMed: 37819745
DOI: 10.1167/iovs.64.13.15 -
Platelets Dec 2023Proteomics tools provide a powerful means to identify, detect, and quantify protein-related details in studies of platelet phenotype and function. Here, we consider how...
Proteomics tools provide a powerful means to identify, detect, and quantify protein-related details in studies of platelet phenotype and function. Here, we consider how historical and recent advances in proteomics approaches have informed our understanding of platelet biology, and, how proteomics tools can be used going forward to advance studies of platelets. It is now apparent that the platelet proteome is comprised of thousands of different proteins, where specific changes in platelet protein systems can accompany alterations in platelet function in health and disease. Going forward, many challenges remain in how to best carry out, validate and interpret platelet proteomics experiments. Future studies of platelet protein post-translational modifications such as glycosylation, or studies that take advantage of single cell proteomics and top-down proteomics methods all represent areas of interest to profiling and more richly understanding platelets in human wellness and disease.
Topics: Humans; Blood Platelets; Proteomics; Phenotype; Proteome
PubMed: 37246523
DOI: 10.1080/09537104.2023.2217932 -
Frontiers in Cellular and Infection... 2022Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in...
BACKGROUND
Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in several studies for their ability to reflect molecular changes caused by SARS-CoV-2. Here we provide insights into the roles of EVs in pathological processes associated with the progression and severity of COVID-19.
METHODS
In this study, we used a label-free shotgun proteomic approach to identify and quantify alterations in EV protein abundance in severe COVID-19 patients. We isolated plasma extracellular vesicles from healthy donors and patients with severe COVID-19 by size exclusion chromatography (SEC). Then, flow cytometry was performed to assess the origin of EVs and to investigate the presence of circulating procoagulant EVs in COVID-19 patients. A total protein extraction was performed, and samples were analyzed by nLC-MS/MS in a Q-Exactive HF-X. Finally, computational analysis was applied to signify biological processes related to disease pathogenesis.
RESULTS
We report significant changes in the proteome of EVs from patients with severe COVID-19. Flow cytometry experiments indicated an increase in total circulating EVs and with tissue factor (TF) dependent procoagulant activity. Differentially expressed proteins in the disease groups were associated with complement and coagulation cascades, platelet degranulation, and acute inflammatory response.
CONCLUSIONS
The proteomic data reinforce the changes in the proteome of extracellular vesicles from patients infected with SARS-CoV-2 and suggest a role for EVs in severe COVID-19.
Topics: COVID-19; Extracellular Vesicles; Humans; Proteome; Proteomics; SARS-CoV-2; Tandem Mass Spectrometry
PubMed: 35937696
DOI: 10.3389/fcimb.2022.926352 -
Molecular & Cellular Proteomics : MCP Jun 2023Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific...
Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).
Topics: Animals; Mice; Microglia; Proteome; Biotin; Proteomics; Lipopolysaccharides; Cell Line; Neurons; Biotinylation
PubMed: 37061046
DOI: 10.1016/j.mcpro.2023.100546 -
Biochemical and Biophysical Research... Oct 2022The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and...
The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and dephosphorylation of neuronal proteins are regulated by synaptic plasticity, no systematic analyses of this have yet been conducted. We performed a phosphoproteomic analysis of hippocampal synaptic plasticity using a nano-Acquity/Synapt LC-MS/MS system. Neuronal proteins were extracted from hippocampal tissues and cultured neurons exposed to long-term potentiation (LTP) or long-term depression (LTD). Filter-aided sample preparation (FASP) was performed to remove residual anionic detergents for complete tryptic digestion. Phosphopeptides were then enriched using TiO chromatography, followed by immunoaffinity chromatography with an anti-phosphotyrosine antibody. Among the 1500 phosphopeptides identified by LC-MS/MS, 374 phosphopeptides were detected simultaneously in both hippocampal tissues and cultured neurons. Semi-quantification counting the number of spectra of each phosphopeptide showed that 42 of 374 phosphopeptides changed significantly depending on synaptic plasticity. In conclusion, a new proteomic method using sequential enrichment of phosphopeptides and semi-quantification enabled the phosphoproteomic analysis of hippocampal synaptic plasticity.
Topics: Chromatography, Liquid; Hippocampus; Long-Term Synaptic Depression; Neuronal Plasticity; Phosphopeptides; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 35981422
DOI: 10.1016/j.bbrc.2022.07.051 -
Briefings in Bioinformatics Jan 2021Empowered by the advancement of high-throughput bio technologies, recent research on body-fluid proteomes has led to the discoveries of numerous novel disease biomarkers... (Review)
Review
Empowered by the advancement of high-throughput bio technologies, recent research on body-fluid proteomes has led to the discoveries of numerous novel disease biomarkers and therapeutic drugs. In the meantime, a tremendous progress in disclosing the body-fluid proteomes was made, resulting in a collection of over 15 000 different proteins detected in major human body fluids. However, common challenges remain with current proteomics technologies about how to effectively handle the large variety of protein modifications in those fluids. To this end, computational effort utilizing statistical and machine-learning approaches has shown early successes in identifying biomarker proteins in specific human diseases. In this article, we first summarized the experimental progresses using a combination of conventional and high-throughput technologies, along with the major discoveries, and focused on current research status of 16 types of body-fluid proteins. Next, the emerging computational work on protein prediction based on support vector machine, ranking algorithm, and protein-protein interaction network were also surveyed, followed by algorithm and application discussion. At last, we discuss additional critical concerns about these topics and close the review by providing future perspectives especially toward the realization of clinical disease biomarker discovery.
Topics: Biomarkers; Body Fluids; Humans; Proteome; Proteomics
PubMed: 32020158
DOI: 10.1093/bib/bbz160 -
Molecular & Cellular Proteomics : MCP Jul 2020Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the... (Review)
Review
Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the development of powerful spatial proteomics approaches for determining protein localization at the whole cell scale. Here, a brief overview of current methods is presented, followed by a detailed discussion of organellar mapping through proteomic profiling. This relatively simple yet flexible approach is rapidly gaining popularity, because of its ability to capture the localizations of thousands of proteins in a single experiment. It can be used to generate high-resolution cell maps, and as a tool for monitoring protein localization dynamics. This review highlights the strengths and limitations of the approach and provides guidance to designing and interpreting profiling experiments.
Topics: Animals; Cell Fractionation; Humans; Mass Spectrometry; Organelles; Protein Interaction Mapping; Protein Transport; Proteome; Proteomics; Spatio-Temporal Analysis; Subcellular Fractions
PubMed: 32345598
DOI: 10.1074/mcp.R120.001971