Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue.
Ontology highlight
ABSTRACT:
SUBMITTER: Murray CI
PROVIDER: S-EPMC2761665 | biostudies-literature | 2009 Aug
REPOSITORIES: biostudies-literature
Publications
Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue.
Murray Christopher I CI Barrett Meagan M Van Eyk Jennifer E JE
Proteomics 20090801 15
The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a c ...[more]
Similar Datasets
Project description:Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI(N)) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI(N) values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.
| S-EPMC3164751 | biostudies-literature
Project description:Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.
| S-EPMC7447005 | biostudies-literature
Project description:We isolated whole embryo, nuclear, and cytoplasmic lysates from Drosophila embryos during early and late embryogenesis In Drosophila melanogaster, cell-type specification during early nervous system development requires precise regulation of gene expression in time and space. To investigate the gene regulation patterns in early neuronal development, we developed a method 'DIV-SortSeq' to enrich for specific neuroglial cell types for RNA sequencing: from early columnar subdivision and specification, through neuroglial differentiation. DIV-SortSeq recapitulates many of the known protein-coding transcriptome dynamics, as well as novel transcriptional regulatory patterns. We also performed nuclear-cytoplasmic fractionation of whole embryos - Fractionation-Seq - to assess temporal changes in subcellular localization of transcripts during embryogenesis. We present these data as a resource to permit further in-depth investigation into the functional roles of the coding and noncoding transcriptome during early Drosophila neurogenesis.
2019-02-19 | GSE106094 | GEO
Project description:Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.
| S-EPMC3618666 | biostudies-literature
Project description:MotivationPathway enrichment analysis has become a key tool for biomedical researchers to gain insight into the underlying biology of differentially expressed genes, proteins and metabolites. It reduces complexity and provides a system-level view of changes in cellular activity in response to treatments and/or in disease states. Methods that use existing pathway network information have been shown to outperform simpler methods that only take into account pathway membership. However, despite significant progress in understanding the association amongst members of biological pathways, and expansion of data bases containing information about interactions of biomolecules, the existing network information may be incomplete or inaccurate and is not cell-type or disease condition-specific.ResultsWe propose a constrained network estimation framework that combines network estimation based on cell- and condition-specific high-dimensional Omics data with interaction information from existing data bases. The resulting pathway topology information is subsequently used to provide a framework for simultaneous testing of differences in expression levels of pathway members, as well as their interactions. We study the asymptotic properties of the proposed network estimator and the test for pathway enrichment, and investigate its small sample performance in simulated and real data settings.Availability and implementationThe proposed method has been implemented in the R-package netgsa available on CRAN.Contactjinma@upenn.eduSupplementary information: Supplementary data are available at Bioinformatics online.
| S-EPMC5939912 | biostudies-literature
Project description:Although compartmentation is a key feature of eukaryotic cells, biological research is frequently limited by methods allowing for the comprehensive subcellular resolution of the metabolome. It has been widely accepted that such a resolution would be necessary in order to approximate cellular biochemistry and metabolic regulation, yet technical challenges still limit both the reproducible subcellular fractionation and the sample throughput being necessary for a statistically robust analysis. Here, we present a method and a detailed protocol which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular localization. The presented benchtop method aims at unraveling subcellular metabolome dynamics in a precise and statistically robust manner using a relatively small amount of tissue material. The method is based on the separation of cellular fractions via density gradients consisting of organic, non-aqueous solvents. By determining the relative distribution of compartment-specific marker enzymes together with metabolite profiles over the density gradient it is possible to estimate compartment-specific metabolite concentrations by correlation. To support this correlation analysis, a spreadsheet is provided executing a calculation algorithm to determine the distribution of metabolites over subcellular compartments. The calculation algorithm performs correlation of marker enzyme activity and metabolite abundance accounting for technical errors, reproducibility and the resulting error propagation. The method was developed, tested and validated in three natural accessions of Arabidopsis thaliana showing different ability to acclimate to low temperature. Particularly, amino acids were strongly shuffled between subcellular compartments in a cold-sensitive accession while a cold-tolerant accession was characterized by a stable subcellular metabolic homeostasis. Finally, we conclude that subcellular metabolome analysis is essential to unambiguously unravel regulatory strategies being involved in plant-environment interactions.
| S-EPMC5177628 | biostudies-literature
Project description:Resulting from the nuclear fuel cycle, large amounts of depleted uranium (DU) tails are piling up, waiting for possible use or final disposal. To date, the recovery of the residual 235U isotope contained in DU has been conducted only marginally by physical processes. Relative isotope abundances are often mediated by biological processes, and the biologically driven U isotopic fractionation has been previously identified in reducing bacteria. Our results indicate that the cells of two microalgal strains (freshwater Chlamydomonas sp. (ChlGS) and marine Tetraselmis mediterranea (TmmRU)) took up DU from the exposure solutions, inducing U isotopic fractionation with a preference for the fissile 235U isotope over 238U. The n(235U)/n(238U) isotopic fractionation magnitudes (?235) were 23.6?±?12.5‰ and 370.4?±?103.9‰, respectively. These results open up new perspectives on the re-enrichment of DU tailings, offering a potential biological alternative to obtain reprocessed natural-equivalent uranium. Additionally, the findings present implications for identifying biological signatures in the geologic records.
| S-EPMC6374374 | biostudies-literature
Project description:The biomechanical properties of populations of diseased cells are shown to have differences from healthy populations of cells, yet the overlap of these biomechanical properties can limit their use in disease cell enrichment and detection. We report a new microfluidic cell enrichment technology that continuously fractionates cells through differences in biomechanical properties, resulting in highly pure cellular subpopulations. Cell fractionation is achieved in a microfluidic channel with an array of diagonal ridges that are designed to segregate biomechanically distinct cells to different locations in the channel. Due to the imposition of elastic and viscous forces during cellular compression, which are a function of cell biomechanical properties including size and viscoelasticity, larger, stiffer and less viscos cells migrate parallel to the diagonal ridges and exhibit positive lateral displacement. On the other hand, smaller, softer and more viscous cells migrate perpendicular to the diagonal ridges due to circulatory flow induced by the ridges and result in negative lateral displacement. Multiple outlets are then utilized to collect cells with finer gradation of differences in cell biomechanical properties. The result is that cell fractionation dramatically improves cell separation efficiency compared to binary outputs and enables the measurement of subtle biomechanical differences within a single cell type. As a proof-of-concept demonstration, we mix two different leukemia cell lines (K562 and HL60) and utilize cell fractionation to achieve over 45-fold enhancement of cell populations, with high purity cellular enrichment (90% to 99%) of each cell line. In addition, we demonstrate cell fractionation of a single cell type (K562 cells) into subpopulations and characterize the variations of biomechanical properties of the separated cells with atomic force microscopy. These results will be beneficial to obtaining label-free separation of cellular mixtures, or to better investigate the origins of biomechanical differences in a single cell type.
| S-EPMC5354170 | biostudies-literature
Project description:The poly(A) tail appended to the 3’ end of many eukaryotic transcripts plays a key role in the stability, nuclear transport, and translation of the RNA. These roles are largely mediated by the Poly(A) Binding Proteins (PABPs) that coat the poly(A) tails and interact with various proteins involved in the biogenesis and function of the RNA. While it is well established that the nuclear PABP (PABPN) binds newly synthesized poly(A) tails and is apparently replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these distinct PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A) tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A) tails and PABPC-enriched RNAs tended to have longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A) tail synthesis, maintenance and function.
2022-03-30 | GSE195493 | GEO
Project description:Retinal degeneration slow (Rds) is a photoreceptor-specific tetraspanin glycoprotein essential for photoreceptor outer segment (OS) morphogenesis. Over 80 mutations in this protein are associated with several different retinal diseases. Rds forms a mixture of disulfide-linked homomeric dimers, octamers, and higher-order oligomers, with Cys150 playing a crucial role in its oligomerization. Rds also forms noncovalent homo- and hetero-tetramers with its nonglycosylated homologue, Rom-1. Here, we evaluated the subcellular site of Rds oligomerization and the pattern of Rds/Rom-1 complex assembly in several types of knockout mice, including rhodopsin (Rho-/-, lacking rod OS), Rom-1 (Rom-1-/-), neural retina leucine zipper (Nrl-/-, cone-dominant), and in comparison with wild-type (WT, rod-dominant) mice. Oligomerization and the pattern of complex assembly were also evaluated in OS-enriched vs OS-depleted preparations from WT and Rom-1-/- retinas. Velocity sedimentation under reducing- and nonreducing conditions and co-immunoprecipitation experiments showed the presence of Rds mainly as homo- and hetero-tetramers with Rom-1 in the photoreceptor inner segment (IS), while higher-order, disulfide-linked intermediate complexes and oligomers were exclusively present in the photoreceptor OS. Rom-1-independent oligomerization of Rds was observed in Rom-1-/- retinas. The pattern of Rds complexes in cones from Nrl-/- mice was comparable to that in rods from WT mice. On the basis of these findings, we propose that Rds traffics from the IS to the OS as homo- and hetero-tetramers, with subsequent disulfide-linked oligomerization occurring concomitant with OS disc morphogenesis (at either the base of OS or the tip of the connecting cilium). These results suggest that Rds mutations that interfere with tetramer formation can block Rds trafficking to the OS, leading to loss-of-function defects.
| S-EPMC2788309 | biostudies-literature