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Protein identification in 1000 U2OS cells

ABSTRACT: In this study, we aim to optimize sample processing methods using a conventional in-solution proteomic sample processing workflow. We then compared protein idetnfication numbers withour improved sample processing methods versus the original method, in 1000 U2OS human osteosarcoma cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Yu Lu

LAB HEAD: Yu Lu

PROVIDER: PXD013702 | Pride | 2019-05-06

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	U2OS-100ng-75min-1.msf	Msf
	U2OS-100ng-75min-1.raw	Raw
	U2OS-100ng-75min-2.msf	Msf
	U2OS-100ng-75min-2.raw	Raw
	U2OS-100ng-75min-3.msf	Msf

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Publications

Step-Wise Assessment and Optimization of Sample Handling Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells.

Wu Ruilin R Xing Sansi S Badv Maryam M Didar Tohid F TF Lu Yu Y

Analytical chemistry 20190801 16

Protein and peptide adhesion is a major factor contributing to sample loss during proteomic sample preparation workflows. Sample loss often has detrimental effects on the quality of proteomic analysis by compromising protein identification and data reproducibility. When starting with a low sample amount, only the most abundant proteins can be identified, which often offers little insights for biological research. Although the general idea about severe sample loss from low amount of starting mate ...[more]

PMID: 31318197

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Influenza infection rewires energy metabolism and induces browning features in adipose cells and tissues

Project description:Influenza A viruses cause epidemics and pandemics with damaging health and economic impacts. Alike any obligate intracellular pathogen, IAVs hijack host cell machinery and energetic resources to multiply within, and eventually exit, the host. Increased fatty acid and cholesterol synthesis, as well as increased glucose metabolism have been identified as the major metabolic changes induced by infection. Besides these effects on metabolism, IAV infection also triggers a variety of innate defense mechanisms within the host cell. Although mainly defined as a virus of the respiratory tract, with airway epithelial cells being its prime cellular habitat, complications outside the site of infection have also been reported. However, whether influenza on its own may impact on endocrine tissues and, thereby, lead to metabolic complications, has never been investigated. Here, we compared the response of preadipocytes and adipocytes to IAV infection, in terms of transcriptomic profiles and bioenergetics. The results showed that IAV triggers a browning adipogenesis process, leading to metabolic reprogramming of the adipose tissue resulting in long-lasting alterations of body metabolism. We conclude that the adipose tissue might be an undervalued organ in influenza pathophysiology.

2019-07-20 | E-MTAB-6646 | biostudies-arrayexpress

Transcription profiling of pig granulosa cells after treatment with follicle-stimulating hormone to find genes potentially important in oocyte maturation and development

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2010-03-31 | E-MEXP-1763 | biostudies-arrayexpress

Proteomics of Metronidazole Resistance in three seminal lines of Giardia duodenalis

Project description:Metronidazole (Mtz) is the frontline chemotherapeutic for a variety of anaerobic pathogens, including the gastroenterinal parasitic protist, Giardia duodenalis. However, treatment failure and clinical resistance is common and increasing. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in in vitro, isogenic cultures. These mechanisms centrally involve regulation of glycolytic and antioxidant oxidoreducatases, however this is inconsistent across isolate lines, likely incomplete, and further complicated by phenotypic plasticity in Giardia, which regains Mtz susceptibility after cessation of drug selection, or via passage through the life cycle. Global, quantitative approaches are required to reconstruct complex Mtz resistance phenotypes, resolve isolate variability, and explore unresolved hypotheses regarding post-translational modifications as the regulators of this phenotype. Our study addresses these gaps by comprehensively characterising proteomic changes in three seminal Mtz resistant lines compared to their isogenic, Mtz-susceptible, parental lines. Using quantitative proteomics, we have calculated differentially expressed proteins (DEPs) in MtzR lines, and probed via Western blot changes in protein acetylation, methylation, ubiquitination and phosphorylation post-translational modifications (PTMs). Finally, we have also performed the first controlled, longitudinal study

2018-02-21 | PXD007183 | Pride

SWATH mass spectrometry performance using extended ion assay libraries

Project description:The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed using peptide MS/MS reference ion assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from shared data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally-generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis we developed a software workflow, SwathXtend, which generates extended peptide assay libraries using a local seed library and delivers statistical analysis of SWATH-based sample comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at different ratios to simulate common protein abundance change comparisons. SWATH-MS data with 2, 5 and 10% of yeast peptides spiked into the human cell lysate were assessed using several local and repository-based assay libraries of different complexities and proteome compositions. We evaluated detection specificity and accuracy to detect differentially abundant proteins and reporting thresholds for statistical analyses. We demonstrate that extended assay libraries integrated with local seed libraries achieve better performance than local limited assay libraries alone from the aspects of the number of peptides and proteins identified and the specificity to detect differentially abundant proteins; the performance of extended assay libraries heavily depend on the similarity of the seed and add-on libraries; statistical analysis with multiple testing correction can improve the statistical rigor needed when using large, extended assay libraries.

2016-05-12 | PXD002836 | Pride

Investigation of the host response of naïve Atlantic salmon (Salmo salar) inoculated with Paramoeba perurans prior to the onset of clinical symptoms

Project description:Amoebic Gill Disease (AGD), caused by the ectoparasite Paramoeba perurans (P. perurans) is characterised by hyperplasia of the gill epithelium and lamellar fusion and has become recognised as one of the most significant health threats in salmon farming . In this study, the gill and serum proteomes of Atlantic salmon inoculated with P. perurans, across multiple timepoints post-challenge, were analysed. The expression of proteins with established roles in innate immunity, across various timepoints, was compared with expression in naïve Atlantic salmon to elucidate the host response to gill colonisation.

2021-04-16 | PXD022101 | Pride

Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability [sequence data]

Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing. HuR (ELAVL1) PAR-CLIP

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Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells - secreted proteins of dexamethasone-treated inflammatory stimulated cells

Project description:Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced up-regulation of proteins such as IL-1beta, IL-6, CXCL2 and GROα was confirmed, however with strong quantitative inter-individual differences. Furthermore, the inability of dexamethasone to down-regulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. While most consequences of dexamethasone were found compatible with the expected way of action, some unexpected but significant observations may relate with adverse effects.

2014-11-04 | PXD001423 | Pride

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Native Prohibitin Complex Enrichment

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