Proteomics quality and standard: from a regulatory perspective.
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ABSTRACT:
SUBMITTER: Gu Q
PROVIDER: S-EPMC8463138 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cultured mouse lymphoma cells were lysed with a buffer containing 50 mM Tris-HCl (pH 8.0) and 8 M Urea with or without a protease inhibitor cocktail (Sigma). The lysates were desalted and digested with sequencing-grade modified trypsin (Promega). Different amounts of peptides were analyzed using nanoflow RPLC-MS/MS on a linear ion trap mass spectrometer (LTQ XL, Thermo Electron, San Jose, CA). The raw MS/MS data were searched using SEQUEST running under BioWorks (Rev. 3.3.1 SP1) (Thermo Electron, San Jose, CA) against a mouse IPI proteome database (Version 3.62) downloaded from the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk). For the SEQUEST analysis, the peptide mass tolerance was set as 2.0 Da and the fragment ion tolerance was 1.0 Da. A tryptic enzyme restriction with a maximum of two internal missed cleavage sites was used. The Xcorr versus charge state values were filtered at Xcorr ≥ 1.7 for [M+H]1+ ions, ≥ 2.5 for [M+2H]2+ ions, ≥ 3.2 for [M+3H]3+ ions, and P ≤ 0.01 and ΔCn ≥ 0.08 were applied for identification of all fully tryptic peptides. The peptide ion chromatogram was extracted using a minimum intensity threshold of 100, mass tolerance of 2.0 amu and smoothing point of 5, and the area of the extracted ion chromatographic (XIC) peak was integrated and calculated using the PepQuan module in Bioworks (Thermo Electron, San Jose, CA).
2022-02-15 | PXD000334 | Pride
Project description:Proteomics based on high-resolution mass spectrometry (MS) is the tool of choice for the analysis of protein presence, modifications and interactions, with increasing emphasis on the examination of tumor tissues. Application of MS-based proteomics offers a detailed picture of tumor tissue characteristics, facilitating the appreciation of different tumor entities, whilst providing reliable and fast results for therapeutic marker targeting and prognostic factor assessment. Through use of the high analytical resolution of nano-high-pressure liquid chromatography (nanoHPLC) and the high resolution of an Orbitrap Elite mass spectrometer, the present study aimed to provide knowledge on the proteome of the generally unknown entity of pediatric ependymal tumors.Ten resected specimens of childhood ependymoma were analyzed through a one-dimensional (1D) nanoLC-MS/MS approach. Method optimization steps were undertaken for both the sample preparation/protein extraction procedure and LC parameters, aiming to achieve the highest possible identification rates.Following method optimization, each nanoLC-MS/MS run resulted in identification of more than 5,000 proteins and more than 25,000 peptides for every analyzed sample, thus detailing the greater part of the ependymoma proteome. Identified proteins were found to spread throughout all known tumor categories regarding their molecular function and subcellular localization.Through the proposed nanoLC-MS/MS method herein we report, for the firs time, the ependymoma proteome database. A large number of similarities regarding proteome content are revealed compared to other two pediatric brain tumor entities; astrocytomas and medulloblastomas. Furthermore, through our approach, the majority of currently proposed markers for ependymoma (e.g. nucleolin, nestin, Ki67 and laminin subunit A2) as well as all major key players of the phosphoinositide 3-kinase pathway (seemingly implicated in ependymoma), were definitely detected.
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Project description:The number of publications in the field of nanogenotoxicology and the amount of genotoxicity data on nanomaterials (NMs) in several databases generated by European Union (EU) funded projects have increased during the last decade. In parallel, large research efforts have contributed to both our understanding of key physico-chemical (PC) parameters regarding NM characterization as well as the limitations of toxicological assays originally designed for soluble chemicals. Hence, it is becoming increasingly clear that not all of these data are reliable or relevant from the regulatory perspective. The aim of this systematic review is to investigate the extent of studies on genotoxicity of NMs that can be considered reliable and relevant by current standards and bring focus to what is needed for a study to be useful from the regulatory point of view. Due to the vast number of studies available, we chose to limit our search to two large groups, which have raised substantial interest in recent years: nanofibers (including nanotubes) and metal-containing nanoparticles. Focusing on peer-reviewed publications, we evaluated the completeness of PC characterization of the tested NMs, documentation of the model system, study design, and results according to the quality assessment approach developed in the EU FP-7 GUIDEnano project. Further, building on recently published recommendations for best practices in nanogenotoxicology research, we created a set of criteria that address assay-specific reliability and relevance for risk assessment purposes. Articles were then reviewed, the qualifying publications discussed, and the most common shortcomings in NM genotoxicity studies highlighted. Moreover, several EU projects under the FP7 and H2020 framework set the aim to collectively feed the information they produced into the eNanoMapper database. As a result, and over the years, the eNanoMapper database has been extended with data of various quality depending on the existing knowledge at the time of entry. These activities are highly relevant since negative results are often not published. Here, we have reviewed the NanoInformaTIX instance under the eNanoMapper database, which hosts data from nine EU initiatives. We evaluated the data quality and the feasibility of use of the data from a regulatory perspective for each experimental entry.
| S-EPMC9472411 | biostudies-literature
Project description:The relationship between data quality and data standards has not been clearly articulated. While some directly state that data standards increase data quality, others claim the opposite. Depending on the type of data standard and the aspects of data quality considered, both arguments may in fact be correct. We deconstruct a typology of data standards and ap ply a dimensional definition of data quality to clearly articulate the relationship between the two, providing a framework for data quality planning.
| S-EPMC4317361 | biostudies-other
Project description:Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.
| S-EPMC5702361 | biostudies-literature
Project description:IntroductionDespite being a leading cause of death worldwide, chronic obstructive pulmonary disease (COPD) is underdiagnosed and underprioritized within healthcare systems. Existing healthcare policies should be revisited to include COPD prevention and management as a global priority. Here, we propose and describe health system quality standard position statements that should be implemented as a consistent standard of care for patients with COPD.MethodsA multidisciplinary group of clinicians with expertise in COPD management together with patient advocates from eight countries participated in a quality standards review meeting convened in April 2021. The principal objective was to achieve consensus on global health system priorities to ensure consistent standards of care for COPD. These quality standard position statements were either evidence-based or reflected the combined views of the panel.ResultsOn the basis of discussions, the experts adopted five quality standard position statements, including the rationale for their inclusion, supporting clinical evidence, and essential criteria for quality metrics. These quality standard position statements emphasize the core elements of COPD care, including (1) diagnosis, (2) adequate patient and caregiver education, (3) access to medical and nonmedical treatments aligned with the latest evidence-based recommendations and appropriate management by a respiratory specialist when required, (4) appropriate management of acute COPD exacerbations, and (5) regular patient and caregiver follow-up for care plan reviews.ConclusionsThese practical quality standards may be applicable to and implemented at both local and national levels. While universally applicable to the core elements of appropriate COPD care, they can be adapted to consider differences in healthcare resources and priorities, organizational structure, and care delivery capabilities of individual healthcare systems. We encourage the adoption of these global quality standards by policymakers and healthcare practitioners alike to inform national and regional health system policy revisions to improve the quality and consistency of COPD care worldwide.
| S-EPMC9047462 | biostudies-literature
Project description:Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid "standard flow" HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.
| S-EPMC8788518 | biostudies-literature
Project description:Bacillus thuringiensis (Bt) is a unique bacterium in that it shares a common place with a number of chemical compounds which are used commercially to control insects important to agriculture and public health. Although other bacteria, including B. popilliae and B. sphaericus, are used as microbial insecticides, their spectrum of insecticidal activity is quite limited compared to Bt. Importantly, Bt is safe for humans and is the most widely used environmentally compatible biopesticide worldwide. Furthermore, insecticidal Bt genes have been incorporated into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture.This review highlights what the authors consider the most relevant issues and topics pertaining to the genomics and proteomics of Bt. At least one of the authors (L.A.B.) has spent most of his professional life studying different aspects of this bacterium with the goal in mind of determining the mechanism(s) by which it kills insects. The other authors have a much shorter experience with Bt but their intellect and personal insight have greatly enriched our understanding of what makes Bt distinctive in the microbial world. Obviously, there is personal interest and bias reflected in this article notwithstanding oversight of a number of published studies. This review contains some material not published elsewhere although several ideas and concepts were developed from a broad base of scientific literature up to 2010.
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Project description:<h4><strong>BACKGROUND: </strong>Semen quality is negatively correlated with male age and is mainly quantified by a routine semen analysis, which is descriptive and inconclusive. Sperm proteins or semen metabolites are used as the intermediate or end-products, reflecting changes in semen quality, and hold much promise as a new biomarker to predict fertility in advanced-aged males.</h4><h4><strong>OBJECTIVES: </strong>In this study, we sought to assess whether the semen metabolome and proteome of aged males can affect semen quality and serve as biomarkers for predicting semen quality.</h4><p><strong>MATERIALS AND METHODS: </strong>We retrospectively analyzed 12825 males that underwent semen routine analysis to understand the age-dependent changes in sperm quality. To identify the difference between aged and young adults, metabolomics (n=60) analyses of semen and proteomics (n=12) analyses of sperm were conducted. Finally, integrated machine learning of metabolomics was conducted to screen biomarkers to identify aging semen.</p><p><strong>RESULTS:</strong> We discovered that male age was positively correlated with sperm concentration as well as DNA fragmentation index(DFI), and negatively with progressive motile sperm count, total sperm count, sperm volume and progressive sperm motility. The differential metabolites were significantly enriched in various metabolic pathways, and four of these differential metabolites (Pipamperone, 2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, Arg-Pro and Triethyl phosphate) were utilized to establish a biomarker panel to identify aging semen. Proteomic analysis showed that differential proteins were significantly enriched in protein digestion and absorption and some energy-related pathways. An integrated analysis of the metabolome and proteome identified differential energy metabolism and oxidative stress-related proteins, which could explain the decreased motility and the increased DFI of aging sperm.</p><p><strong>DISCUSSION AND CONCLUSION:</strong> We provide compelling evidence that the changes in semen metabolome and sperm proteome are related to the decline of semen quality in aged males. Moreover, a biomarker panel based on four metabolites was established to identify aging semen.</p>
2024-01-16 | MTBLS5289 | MetaboLights
Project description:The original PRIDE Inspector tool was developed as an open source standalone tool to enable the visualization and validation of mass-spectrometry (MS)-based proteomics data before data submission or already publicly available in the Proteomics Identifications (PRIDE) database. The initial implementation of the tool focused on visualizing PRIDE data by supporting the PRIDE XML format and a direct access to private (password protected) and public experiments in PRIDE.The ProteomeXchange (PX) Consortium has been set up to enable a better integration of existing public proteomics repositories, maximizing its benefit to the scientific community through the implementation of standard submission and dissemination pipelines. Within the Consortium, PRIDE is focused on supporting submissions of tandem MS data. The increasing use and popularity of the new Proteomics Standards Initiative (PSI) data standards such as mzIdentML and mzTab, and the diversity of workflows supported by the PX resources, prompted us to design and implement a new suite of algorithms and libraries that would build upon the success of the original PRIDE Inspector and would enable users to visualize and validate PX "complete" submissions. The PRIDE Inspector Toolsuite supports the handling and visualization of different experimental output files, ranging from spectra (mzML, mzXML, and the most popular peak lists formats) and peptide and protein identification results (mzIdentML, PRIDE XML, mzTab) to quantification data (mzTab, PRIDE XML), using a modular and extensible set of open-source, cross-platform libraries. We believe that the PRIDE Inspector Toolsuite represents a milestone in the visualization and quality assessment of proteomics data. It is freely available at http://github.com/PRIDE-Toolsuite/.
| S-EPMC4762524 | biostudies-literature