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Comparison of in-solution and SP3 sample processing workflows on HeLa cell samples

ABSTRACT: Comparison of in-solution and single-pot, solid-phase-enchanced sample preparation (SP3) sample processing workflows on HeLa cell samples, using different lysis buffers based on SDS and guanidine hydrochloride, for optimal analysis by liquid chromatography mass spectrometry (LC-MS)

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Epithelial Cell, Cell Culture

SUBMITTER: Maria Hernandez-Valladares

LAB HEAD: Frode Berven

PROVIDER: PXD031918 | Pride | 2022-08-12

REPOSITORIES: pride

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			Action	DRS
	Eclipse_091221_IsD_Gua_2_B.msf	Msf
	Eclipse_091221_IsD_Gua_2_B51.raw	Raw
	Eclipse_091221_IsD_Gua_2_B61.raw	Raw
	Eclipse_091221_IsD_Gua_2_B71.raw	Raw
	Eclipse_091221_IsD_Gua_2_B81.raw	Raw

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Publications

Comparing Efficiency of Lysis Buffer Solutions and Sample Preparation Methods for Liquid Chromatography-Mass Spectrometry Analysis of Human Cells and Plasma.

Neset Lasse L Takayidza Gracious G Berven Frode S FS Hernandez-Valladares Maria M

Molecules (Basel, Switzerland) 20220525 11

The use of a proper sample processing methodology for maximum proteome coverage and high-quality quantitative data is an important choice to make before initiating a liquid chromatography-mass spectrometry (LC-MS)-based proteomics study. Popular sample processing workflows for proteomics involve in-solution proteome digestion and single-pot, solid-phase-enhanced sample preparation (SP3). We tested them on both HeLa cells and human plasma samples, using lysis buffers containing SDS, or guanidiniu ...[more]

PMID: 35684327

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Three protein extraction methods for mass spectrometry proteomics of fresh frozen mouse tissue

Project description:We present three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.

2020-07-30 | PXD020256 | Pride

Plasma samples processed with depletion and non depletion

Project description:Plasma samples processed with no depletion and most-abundant-proteins depletion procedures according to standard in-solution and single-pot, solid-phase-enhaced sample preparation (SP3) workflows using guanidyne hydrochloride and SDS lysis buffers for analysis with liquid chromatography mass spectrometry (LC-MS).

2022-08-12 | PXD031919 | Pride

Gene expression profiling of E12.5 wildtype- and Sp3 null hearts

Project description:Mice lacking the zinc finger transcription factor Specificity protein 3 (Sp3) die prenatally in the C57Bl/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at E10.5, and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analysed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of Cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. ChIP analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development, and suggest that it has a crucial role in myocardial differentiation. ( Keywords: Transcription factors, Sp3, knockout mice, cardiac malformations, E12.5

2007-11-01 | GSE9124 | GEO

siRNA knockdown of Sp3 in human

Project description:Multiple sclerosis (MS) is an inflammatory disease of the central nervous system and is generally considered to be autoimmune in nature. We previously demonstrated that the transcription factor Sp3 is significantly down-regulated in immune cells from MS patients. The potential role of Sp3 down-regulation in MS pathogenesis is not well understood. The function of endogenous Sp3 was assessed in vitro after siRNA-mediated knockdown of its transcript in Jurkat cells. Sp3 protein levels were reduced an average of 70%. ELISA studies demonstrated decreased endogenous production of IL-10 and TGFÎ²1 and increased endogenous production of TNFÎ± (p<0.05 in all assays). Subsequent microarray analysis demonstrated significantly altered expression of 36 genes (p<0.001 for each gene) compared with control samples. Analysis showed differential expression (p<0.005) of 8 gene pathways. Many of the genes and pathways that were regulated by Sp3 are involved in immune function, specifically with regard to apoptosis, cell-to-cell adhesion, integrin signaling, T-cell differentiation, and cytokine production. This study identifies mechanisms by which Sp3 may regulate immune function and suggests a basis for its potential contribution to MS disease pathogenesis. Experiment Overall Design: Two Sp3 siRNA knockdown groups (siRNA Sp3 #2: n=3, and #6: n=3) were clustered together into the Treated condition, and the two control groups (siRNA for GAPDH: n=3, and Non-transfected cells: n=3)were assembled to constitute the Control condition. Thus a total of 12 arrays (6 Controls and 6 treated) were used in this experiment.

2008-04-07 | E-GEOD-7102 | biostudies-arrayexpress

Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads

Project description:Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger-scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief (5-minute) centrifugation—either with or without low-cost inert glass beads—as a means of acetonitrile-induced aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1–5000µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling revealed SP4 yielded greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80% vs. 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs, across eight sample types, and five lysis buffers—all confirming equivalent or improved proteome characterisation vs. SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein clean-up, and identifies that magnetic capture risks losses, especially at higher protein concentrations and amongst more hydrophobic proteins. SP4 offers a minimalistic approach to protein clean-up that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications—all whilst retaining the speed and compatibility of SP3.

2022-08-01 | PXD032095 | Pride

Transcription profiling of mouse heart from E12.5 wildtype- and Sp3 animals

Project description:Mice lacking the zinc finger transcription factor Specificity protein 3 (Sp3) die prenatally in the C57Bl/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at E10.5, and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analysed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of Cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. ChIP analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development, and suggest that it has a crucial role in myocardial differentiation. ( Experiment Overall Design: Hearts were dissected from E12.5 wildtype (n=3) and Sp3 knockout (n=3) fetuses. Total RNA was isolated from individual hearts; 5Î¼g was used for labelling and hybridization to 430 2.0 Gene Chips (a total of 6).

2008-06-17 | E-GEOD-9124 | biostudies-arrayexpress

Identification of differential expressed genes after SP1 or SP3 knockdown in colorectal cancer cells

Project description:SP1 and SP3 have been reported to play an critical role in the pregression of tumors. In order to elucidate the molecular mechanism of SP1 and SP3 in colorectal cancer, RNA-seq analysis was performed on SP1 and SP3 knockdown and control HCT116 cells.

2024-07-01 | GSE179314 | GEO

Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development

Project description:Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here we use RNAseq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than WT and Sp+/- littermates, died soon after birth, and had abnormal lung morphogenesis. RNAseq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways, and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development. Development • Accepted manuscript lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways, and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development.

2023-02-28 | GSE225703 | GEO

The transcription factor Sp3 cooperates with HDAC2 to regulate synaptic function and plasticity in neurons

Project description:The histone deacetylase HDAC2, which negatively regulates neuronal plasticity and synaptic gene expression, is upregulated both in Alzheimer’s disease (AD) patients and mouse models (Graff et al., 2012). Therapeutics targeting HDAC2 are speculated to be a promising avenue for ameliorating AD related cognitive impairment. However, attempts to generate HDAC2-specific inhibitors have not been successful. Here, we take a novel approach utilizing integrative genomics to identify proteins that mediate HDAC2 recruitment to synaptic plasticity genes. Functional screening revealed that knockdown of the transcription factor Sp3 phenocopied HDAC2 knockdown, and that Sp3 facilitated the recruitment of HDAC2 to synaptic genes. Importantly, like HDAC2, Sp3 expression was elevated in AD patients and mouse models, where Sp3 knockdown ameliorated synaptic dysfunction. Furthermore, exogenous expression of an HDAC2 fragment containing the Sp3 binding domain fully restored synaptic plasticity and memory in a mouse model with severe neurodegeneration. Our findings indicate that targeting the HDAC2-Sp3 complex could enhance synaptic and cognitive function, without affecting HDAC2 function in other processes.

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siRNA-mediated knockdown of Sp3 induces a pro-inflammatory pattern of cytokine production: relevance to MS

Project description:Multiple sclerosis (MS) is an inflammatory disease of the central nervous system and is generally considered to be autoimmune in nature. We previously demonstrated that the transcription factor Sp3 is significantly down-regulated in immune cells from MS patients. The potential role of Sp3 down-regulation in MS pathogenesis is not well understood. The function of endogenous Sp3 was assessed in vitro after siRNA-mediated knockdown of its transcript in Jurkat cells. Sp3 protein levels were reduced an average of 70%. ELISA studies demonstrated decreased endogenous production of IL-10 and TGFβ1 and increased endogenous production of TNFα (p<0.05 in all assays). Subsequent microarray analysis demonstrated significantly altered expression of 36 genes (p<0.001 for each gene) compared with control samples. Analysis showed differential expression (p<0.005) of 8 gene pathways. Many of the genes and pathways that were regulated by Sp3 are involved in immune function, specifically with regard to apoptosis, cell-to-cell adhesion, integrin signaling, T-cell differentiation, and cytokine production. This study identifies mechanisms by which Sp3 may regulate immune function and suggests a basis for its potential contribution to MS disease pathogenesis. Keywords: siRNA knockdown; Jurkat T-cells; Multiple Sclerosis

2008-02-21 | GSE7102 | GEO

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