Browse
Submit Data
Databases
API
Help

Dataset Information

373 Views

0 Connections

4 Citations

0 Reanalyses

0 Downloads

Omics score: 0

IPG Strip-Based Peptide Fractionation for Shotgun Proteomics

ABSTRACT: This deep proteome dataset of human HEK293 cells was generated using IPG Strip-Based fractionation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Henrik Zauber

LAB HEAD: Prof. Dr. Matthias Selbach

PROVIDER: PXD002389 | Pride | 2015-07-08

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

IPG strip-based peptide fractionation for shotgun proteomics.

Eravci Murat M Sommer Christian C Selbach Matthias M

Methods in molecular biology (Clifton, N.J.) 20140101

Efficient fractionation of peptides is an essential prerequisite for comprehensive analysis of complex protein mixtures by shotgun mass spectrometry. The separation of peptides by isoelectric focusing is particularly attractive due to its orthogonality to reverse-phase HPLC. Here, we present a protocol for in-gel peptide isoelectric focusing using immobilized pH gradient strips. The method shows high resolving power for up to 1 mg of sample and is highly reproducible. ...[more]

PMID: 24791982

Similar Datasets

IPG Strip-Based Peptide Fractionation for Shotgun Proteomics

Project description:This deep proteome dataset of human HEK293 cells was generated using IPG Strip-Based fractionation.

2016-10-21 | MSV000080274 | MassIVE

HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.

Project description:In this project we aimed at improving phosphoproteomics analysis by taking advantage of high resolution isoelectric focusing (HiRIEF) fractionation. We developed a workflow that employs titanium dioxide phospho-enrichment, followed by isobaric labeling with Tandem Mass Tags (TMT) and HiRIEF on a broad pI range (immobilized pH gradient, IPG gel strips employed were 2.5-3.7 and 3-10, Phospho HiRIEF LC-MS). We analyzed HeLa cells untreated (four biological replicates), treated with pervanadate or arrested in mitosis (three biological replicates each). Employing a relatively low amount of material (300 μg of peptides), we identify 22,674 phosphorylation sites, of which 19,036 were localized with high confidence. We demonstrate isoelectric point dependent fractionation of the peptides based on the number of phosphate groups that they carry: 18% of the phospho-peptides identified with the IPG 2.5-3.7 gel strip are multiply phosphorylated peptides and they localize predominantly in the most acidic pI fractions. Identified phosphorylation sites include 1,198 tyrosine phosphorylation sites and 1,491 phospho-sites that were not previously reported in the PhosphoSitePlus database. Total protein quantification performed by standard HiRIEF on the same samples identified 9,185 proteins, of which 4,575 overlap with the proteins identified by Phospho HiRIEF LC-MS. Phosphorylation sites corresponding to these proteins were normalized to total protein abundance, resulting in 18,374 quantified phospho-sites. Kinase association analysis on the quantified phospho-sites resulted in identification of a subset that has putative functions during the mitotic phase and protein-protein interaction network analysis shows a high degree of connectivity of these putatively functional novel phospho-sites.

2017-07-07 | PXD005410 | Pride

Proteomic profiling of glioblastoma stem cells, human astrocyte, and T98G glioblastoma cell line

Project description:With HiRIEF LC-MS/MS shotgun proteomics, we analysed 6 patient-derived glioblastoma stem cells (BT stem cells) and compared them to an astrocyte line (CliniSciences, Guidonia Montecelio, Italy) and a more differentiated glioblastoma cell line (T98G). Each of the 8 cell line sample was run in triplicates in a total of three TMT10 sets, assigning each replicate in a separate TMT10 set, using 2 internal standards per set. The three TMT10 sets were ran in two experiments, first on immobilized pH gradient (IPG) 3-10 isoelectric focusing (IEF) strips, and then on IPG 3.7-4.9 IEF strips.

2022-12-23 | PXD027335 | Pride

Expression data from white adipose tissue of adult mice with mutations in Regulator of sex-limitation genes

Project description:Regulator of sex-limitation (rsl) is a recessive mouse phenotype in which the otherwise male-specific sex-limited protein (Slp) gene is expressed in females. Positional cloning in rsl mice led to the identification of mutations in two neighboring KRAB zinc finger transcriptional repressors, Rsl1 and Rsl2, and BAC transgenic rescue experiements verified their ability to repress male-specific genes in the liver. Microarrays were used here to identify RSL-sensitive genes in mouse abdominal white adipose tissue. Experiment type: sex comparison and genetic modification Total RNA was extracted from abdominal white adipose tissue of adult male and female wild type and Rsl-null mice. Equivalent amounts of RNA were pooled from five mice per sex and genotype. Two pools per sex and genotype were hybridized to the Affymetrix Mouse Genome 430 2.0 PM Array Strip.

2013-07-23 | E-GEOD-49075 | biostudies-arrayexpress

Membrane enriched proteome of transfected SW480 cells

Project description:This project used membrane-enriched proteomics to compare the membrane proteome of SW480 human colorectal cancer subclones that had been transfected with the full length coding sequence of the beta-6 integrin subunit under an overexpression promoter (SW480OE) against SW480 cells that had been transfected with an ‘empty’ mock vector (SW480M). Cell lysate samples were enriched for membrane proteins using a modified sodium carbonate stripping method before undergoing peptide immobilised pH gradient isoelectric focusing (IPG-IEF). Two IPG-IEF pH ranges were used to increase proteomic coverage: pH3-10 (broad range; BR) and pH3.5-4.5 (narrow range; NR). Each IPG-IEF strip was cut into 24 equal fractions and the peptides eluted. IPG-IEF of each pH range for both cell lines was performed in triplicate, resulting in four triplicate sets of 24 fractions. Data processing and bioinformatics: Spectra were identified against the *Homo sapiens* protein database (derived from Ensembl, Swiss-Prot and NCBI) using X!Tandem and the Global Proteome Machine (GPM) Tornado XE (build date 2010.12.01). GPM searches were set to tryptic peptides under GPM’s default fragment ion mass tolerance of 0.4Da and did not include semi-cleavage. Searches included single acid polymorphisms and default GPM residue modifications (oxidation,dioxidation, carbamidomethyl, deamidation, acetylation).

2013-05-30 | PXD000230 | Pride

Human Proteome Dataset Hek293, k652 and HeLa cell lines

Project description:Triple Silac approach to analyze cellline specific protein expression. Hek293 (Heavy, Lys8 and Arg10), K526 (Medium, DMEM with Lys 4 and Arg6), HeLa (Light).

2015-07-08 | PXD002383 | Pride

Vector and Brown fat lncRNA 1 (Blnc1) expressing differentiated brown adipocytes

Project description:Blnc1 is a novel nuclear lncRNA that promotes brown and beige adipocyte differentiation and function. Blnc1 forms a ribonucleoprotein complex with transcription factor EBF2 to stimulate the thermogenic gene program. Further, Blnc1 itself is a target of EBF2, thereby forming a feedforward regulatory loop to drive adipogenesis toward thermogenic phenotype. We used microarrays to elucidate the role of Blnc1 on brown adipocyte differentiation and the induction of the thermogenic gene program. Brown adipocytes expressing vector or brown fat lncRNA 1 (blnc1) were differentiated for 6 days and harvested for RNA isolation and microarray using Affymetrix Mouse MG-430 PM Strip arrays. Two replicated samples were included in this study.

2015-02-28 | E-GEOD-55551 | biostudies-arrayexpress

Scramble and Brown fat lncRNA 1 knockdown (shBlnc1) expressing differentiated brown adipocyte

Project description:Blnc1 is a novel nuclear lncRNA that promotes brown and beige adipocyte differentiation and function. Blnc1 forms a ribonucleoprotein complex with transcription factor EBF2 to stimulate the thermogenic gene program. Further, Blnc1 itself is a target of EBF2, thereby forming a feedforward regulatory loop to drive adipogenesis toward thermogenic phenotype. We used microarrays to elucidate the role of Blnc1 on brown adipocyte differentiation and mitochondrial function. Brown adipocytes expressing Scramble or brown fat lncRNA 1 knockdown (shBlnc1) were differentiated for 6 days and harvested for RNA isolation and microarray using Affymetrix Mouse MG-430 PM Strip arrays. Two replicated samples were included in this study.

2015-02-28 | E-GEOD-57540 | biostudies-arrayexpress

Conservation of mRNA and Protein Expression during Development of C.elegans

Project description:A detailed description of the sample processing is published (Grün et al., 2014 Cell Rep). Briefly, a super Silac Mix using Lys8 was generated and spiked into all samples as an internal reference. Total Lysate was fractionated using SDS-PAGES. Up to 16 fractions were collected per sample. Proteins were in gel digest using LysC only.

2015-07-09 | PXD002482 | Pride

Wild type and Neuregulin 4 (Nrg4) knockout mouse livers

Project description:Nrg4 is a brown fat-enriched adipokine that binds to the liver and preserves metabolic homeostasis during chronic energy excess. Adipose tissue Nrg4 expression is reduced in mouse and human obesity. Nrg4 deficient mice develp more severe insulin resistance and hepatic fat accumulation upon high-fat diet feeding. We used microarrays to elucidate the metabolic pathways that are regulated by Nrg4 in the liver. Wild type and Neuregulin 4 (Nrg4) knockout mice were fed high-fat diet (Research Diets D12492) for eight weeks. Tissues were harvested under ad lib condition for RNA isolation and microarray hybridization using Affymetrix Mouse MG-430 PM Strip arrays. Three independent pools of WT and KO mouse liver RNA were included in the study. Manuscript title: A brown fat-enriched secreted factor preserves metabolic homeostasis through attenuating hepatic lipogenesis, (journal information TBD)

2014-11-17 | E-GEOD-53877 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data