Browse
Submit Data
Databases
API
Help

Dataset Information

595 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

MStern Blot vs. FASP - Different sample types

ABSTRACT: Three different sample types (HeLa lysate, human CSF and human Urine) that were prepared with MStern Blot and FASP. For each sample type there are four replicates.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine, Cerebrospinal Fluid, Cell Culture

SUBMITTER: Sebastian Berger

LAB HEAD: Hanno Steen, PhD

PROVIDER: PXD002572 | Pride | 2015-07-30

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	Data_140702_STB_MStern_CSF-1.mgf	Mgf
	Data_140702_STB_MStern_CSF-1.wiff	Wiff
	Data_140702_STB_MStern_CSF-1.wiff.scan	Wiff
	Data_140702_STB_MStern_CSF-1.xml	Xml
	Data_140702_STB_MStern_CSF-1.zip	Other

Items per page:

1 - 5 of 120

Publications

MStern Blotting-High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates.

Berger Sebastian T ST Ahmed Saima S Muntel Jan J Cuevas Polo Nerea N Bachur Richard R Kentsis Alex A Steen Judith J Steen Hanno H

Molecular & cellular proteomics : MCP 20150729 10

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membra ...[more]

PMID: 26223766

Similar Datasets

Data in support of Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis

Project description:The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiology, prognosis, disease severity, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We use high-density protein array technology to evaluate ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Methods: Pooled plasma samples from 10 anti-CCP negative and 15 anti-CCP positive RA patients were assessed for ACPA-content using a modified protein microarray containing 1,631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PAD) 2 and PAD4. Results: IgG antibodies from anti-CCP positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination for 29 and 26 proteins, respectively. For only 4 proteins, significantly more ACPA-binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for 1 protein.

2022-01-05 | PXD024955 | Pride

Urine Proteomics for Noninvasive Monitoring of Biomarkers in Bronchopulmonary Dysplasia

Project description:Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which can affect the health status of these frail neonate. We tested our hypotheses 1) it is feasible to use early urine samples from extremely low gestational age newborns at risk for bronchopulmonary dysplasia for proteomics, and 2) urine proteomics can confirm previously identified proteins and biomarkers associated with BPD without invasive sample collection. We developed a robust high throughput urine proteomics methodology that requires only 50 microliters of urine. We validated the methodology on urine collected within 72 hours of birth. Urine samples were collected from extremely low gestational age newborns (ELGANS) (gestational age (26 + 1.2) weeks) admitted to a single Neonatal Intensive Care Unit(NICU); half of whom eventually developed BPD, while the other half served as controls. Our high throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes. Interestingly, sixteen identified urinary proteins are known targets of drugs approved by the Food and Drug Administration (FDA). Urine proteomics can be used for prediction of BPD risk. In addition to identifying numerous proteins implicated in BPD pathophysiology, previously found in invasively collected blood, tracheal aspirate, and broncho-alveolar lavage, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine for sequential proteomic evaluations could also allow for longitudinal monitoring of disease progression and impact of therapeutic intervention.

2022-04-04 | PXD028895 | Pride

Quantitative evaluation of FASP and MED FASP protocols

Project description:In this work we re-evaluate FASP and the related Multienzyme digestion (MED)-FASP method. We use different types of animal tissue and cultured cells, and test the performance of the method under various conditions. We analyze the protein-to-peptide conversion by assessing the yield of peptides, frequency of peptides with missed cleavage sites, and the reproducibility of FASP. We identify conditions allowing efficient protein processing with high peptide yields and demonstrate advantages of the two step digestion strategy over single step digestion with trypsin. In addition we show that FASP outperforms in solution cleavage strategies. Our results clearly show that the performance of the digestion varies between different types of samples. We show that MED-FASP in combination of the Total Protein Approach provide highly reproducible protein abundance values. The presented data can be used as guide for optimization of the sample processing

2016-04-28 | PXD003733 | Pride

Comparative evaluation of different sample preparation workflows for reproducible, quantitative, and in-depth analysis of urine proteomics

Project description:The growing field of urinary proteomics has provided a promising opportunity to identify biomarkers useful for diagnosis and prognostication of a number of diseases. Urine is abundant and readily collected in a non-invasive manner which makes it eminently available for testing, however sample preparation for proteomics analysis remains one of the biggest challenges in this field. As newer technologies to analyze tandem mass spectrometry (MS) data develop, utility of urinary proteomics would be enhanced by better sample processing and separation workflows to generate fast, reproducible, and more in-depth proteomics data. In this study, we have evaluated the performance of four sample preparation methods: MStern, PreOmics In-StageTip (iST), Suspension-trapping (S-Trap), and conventional urea In-Solution trypsin hydrolysis for non-depleted urine samples. Data Dependent Acquisition (DDA) mode on QE-HF was used for single-shot label-free data acquisition. Our results demonstrate a high degree of reproducibility within each workflow. PreOmics iST yields the best digestion efficiency with lowest percentage of missed-cleavage peptides. S-trap workflow gave the greatest number of peptide and protein identifications. Using S-trap method, with 0.5mL urine sample as starting material, we identify ~1500 protein groups and ~17700 peptides from DDA analysis with a single injection. The continued refinement of sample preparation (presented here), LC separation methods and mass spectrometry data acquisition can allow the integration of information rich urine proteomic records across large cohorts with other ‘big data’ initiatives becoming more popular in medical research.

2020-03-06 | PXD016273 | Pride

Genome-scale DNA methylation profiling of TEL/AML positive childhood acute lymphoblastic leukaemia

Project description:DNA methylation profiling of 48 samples to determine a DNA methylation signature specific to leukaemic bone marrow samples. Matching Leukaemia bone marrow and day 28 remission bone marrow or post induction follow up (follow up up to 2 years post induction) genomic DNA were extracted from archived microscope smear slides from 11 children diagnosed with TEL/AML positive Acute Lymphoblastic Leukaemia (ALL). Unmatched bone marrow samples from an additional 8 children were also analysed. Primary tissue control samples from CD34+ and CD19+ bone marrow from adult and childhood samples as well as model cell lines (cancer and non cancerous) were compared to primary diseased samples. Disease specific DNA methylation changes were identified from these results and then verified using SEQUENOM EpiTYPER. A total of 48 samples were analysed by Infinium DNA methylation analysis. As per manufacturer's protocols. Replicates were performed on one primary tumour sample and one cell line sample.

2012-12-31 | E-GEOD-29189 | biostudies-arrayexpress

PS2APP sorted cell types

Project description:Mice of indicated genotypes were perfused and their brains dissected and dissociated. Cells were fixed, immunolabeled and FACS sorted. RNA was extracted from neuron, astrocyte, and microglial cell populations. Typical RIN=4-5 for neurons, 6-8 for astrocytes, and 5-7 for microglia. Typical RNA yields 200-300ng for neurons, ~20ng for microglia, and ~5ng for astrocytes. cDNA was generated from up to 25 ng of total RNA using Nugen’s RNA-Seq method for low-input RNA samples, Ovation RNA-Seq System V2 (NuGEN). (Per manufacturers instructions, total RNA was neither depleted of rRNA nor polyA-selected.) 1 μg of sheared cDNA was taken into further processing, starting at end repair step, using Illumina’s TruSeq RNA Sample Preparation Kit v2 (Illumina). The SAMPLE_ID sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0006717

2019-07-25 | GSE129770 | GEO

Phosphorylation of OSBL8 protein in interphase vs mitotic human cells

Project description:Phosphorylation of OSBL8 human protein was compared in interphase vs mitotic Hela cells with a new software developed in-house, Msviz, a graphical software tool developed for in-depth manual validation and quantitation of post-translational modifications.

2017-07-03 | PXD006254 | Pride

Tau-P301L sorted cell types

Project description:Mice of indicated sexes and genotypes were perfused and their brains dissected and dissociated. Cells were fixed, immunolabeled and FACS sorted. RNA was extracted from neuron, astrocyte, and microglial cell populations. Typical RIN=4-5 for neurons, 6-8 for astrocytes, and 5-7 for microglia. Typical RNA yields ~100ng for neurons, ~20ng for microglia, and ~10ng for astrocytes. cDNA was generated from up to 25 ng of total RNA using Nugen’s RNA-Seq method for low-input RNA samples, Ovation RNA-Seq System V2 (NuGEN). (Per manufacturers instructions, total RNA was neither depleted of rRNA nor polyA-selected.) 1 μg of sheared cDNA was taken into further processing, starting at end repair step, using Illumina’s TruSeq RNA Sample Preparation Kit v2 (Illumina). The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0010699

2017-12-20 | GSE93179 | GEO

Tau-P301S sorted cell types

2017-12-20 | GSE93180 | GEO

Near-infrared fluorescent northern blot.

Project description:Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

| S-EPMC6239192 | biostudies-literature