Browse
Submit Data
Databases
API
Help

Dataset Information

332 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

On-bead vs FASP vs InGel sample preparation for LC-MS/MS

ABSTRACT: The aim of this project was to compare and optimize sample processing protocols up-stream of LC-MS/MS. We set out to create a protocol that is easier and less laborious to use with liquid handlers than FASP, but that generates comparable results as FASP or traditional in-gel digestion.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leukocyte, Jurkat Cell

SUBMITTER: Krzysztof Sikorski

LAB HEAD: Fridtjof Lund-Johansen

PROVIDER: PXD005944 | Pride | 2018-07-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	P3617.raw	Raw
	P3619.raw	Raw
	P3621.raw	Raw
	P3623.raw	Raw
	P3625.raw	Raw

Items per page:

1 - 5 of 76

Similar Datasets

MStern Blot vs. FASP - Different sample types

Project description: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.

2015-07-30 | PXD002572 | Pride

Sensitivity and protein digestion course of proteomic Filter Aided Sample Preparation

Project description:1. Sensitivity of FASP was tested using SDS lysates from HeLa cells and mouse brain. Peptides were analyzed using a QExactive HF-X instrument. We found that FASP allows effective processing samples at the low and sub-microgram range. 2. Whole cell lysates of Hela cells were processed with FASP using single or double, consecutive or successive, digestion with LysC or trypsin. The generated peptides were analyzed using a LTQ-Orbitrap mass spectrometer. The results show that short digestion times apparently do not affect the number of identified proteins.

2019-11-12 | PXD014288 | Pride

Identification and quantification of HDA14-dependent substrates in the Arabidopsis thaliana thylakoid acetylome

Project description:Lysine acetylation is a common post-translational modification in eukaryotes and prokaryotes which is known to be involved in the regulation of various cellular processes, such as transcriptional activation, metabolic signaling, and energy homeostasis. Nevertheless, its role in plant primary metabolism, and photosynthesis in particular, is not well understood. Lysine acetylation is catalyzed by acetyltransferases (KATs) and removed by corresponding deacetylases (KDACs). The Arabidopsis thaliana genome encodes for at least 16 KATs and 18 KDACs, belonging to seven different gene families. The cellular functions of these enzymes have been explored only partially, describing effects of single enzymes and their interaction with specific targets. HDA14 is a KDAC expressed primarily in chloroplasts which suggests an involvement in the regulation of photosynthesis or related metabolic processes. In this dataset we aimed to identify substrates of the histone deacetylase 14 in a global manner by comparing the lysine acetylome on leaf thylakoids isolated from hda14 KO plants and wild-type plants, respectively. Proteins were extracted and digested using an adapted FASP procedure, peptides were dimethyl-labeled, and lysine-acetylated peptides were antibody-enriched. Peptide identification and quantitative data analysis was carried out using MaxQuant.

2017-10-25 | PXD006652 | Pride

Ultrasonic-based filter aided sample preparation as the general method to sample preparation in proteomics

Project description:In this work, we propose a new high-throughput ultrafast method for large scale proteomics approaches by speeding the classic filter aided sample preparation protocol, FASP. The new US-FASP method matches the analytical minimalism roles as time, cost, sample requirement, reagent consumption, energy requirements and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all the advantages of the filter aided sample preparation protocol are maintained.

2020-06-08 | PXD018360 | Pride

Urinary proteome of a healthy human cohort aged 70 to 82

Project description:12 urine samples from 6 individuals (2 time points each) enrolled in a study on healthy aging were collected. Samples were frozen at -80⁰C prior to analysis. Samples were thawed, pH neutralized, separated into soluble urine (SU) and urinary pellet (UP) fractions, and subjected to filter aided sample preparation (FASP). FASP-processed samples were subjected to LC-MS/MS analysis on a Q Exactive mass spec.

2017-05-03 | PXD004713 | Pride

Filter-aided sample preparation depends on filter unit shape

Project description:One of the main goals of shotgun proteomics studies is to identify by mass spectrometry as many proteins as possible, often from low-protein samples, in order to help answer important biological questions. All experimental steps need to be optimized to achieve a thorough proteomic analysis. One of the most important is an optimal extraction and solubilization of proteins through the use of detergents, chaotropes, and reducing agents. Although very efficient in solubilizing membrane proteins, the presence of these detergents, such as SDS, negatively affects the LC/MS analysis and limits protein identifications, and therefor they need to be removed before LC-MS analysis. Filter-aided sample preparation (FASP) is a generally accepted method for removal of detergents and chaotropes used for protein extraction as well as non-proteinaceous compounds such as salts, nucleic acids and lipids. Another critical aspect of proteomic analyses is the starting amount of protein in the sample. There is still little information about the efficacy of FASP method for protein-limited samples. The aim of this study was to compare two methods for SDS removal, ethanol precipitation versus FASP and we evaluate the effectiveness of FASP method using different filtration devices, as well as RIPA (0.1% SDS) and high-SDS (2%SDS) lysis buffers applied to low (1µg) and high (10-100µg) protein amounts.

2020-11-05 | PXD003400 | Pride

Identification and quantification of HDA14-dependent substrates in the Arabidopsis thaliana leaf acetylome of plants grown under low-light conditions

Project description:Lysine acetylation is a common post-translational modification in eukaryotes and prokaryotes which is known to be involved in the regulation of various cellular processes, such as transcriptional activation, metabolic signaling, and energy homeostasis. Nevertheless, its role in plant primary metabolism, and photosynthesis in particular, is not well understood. Lysine acetylation is catalyzed by acetyltransferases (KATs) and removed by corresponding deacetylases (KDACs). The Arabidopsis thaliana genome encodes for at least 16 KATs and 18 KDACs, belonging to seven different gene families. The cellular functions of these enzymes have been explored only partially, describing effects of single enzymes and their interaction with specific targets. HDA14 is a KDAC expressed primarily in chloroplasts which suggests an involvement in the regulation of photosynthesis or related metabolic processes. In this dataset we aimed to identify substrates of the histone deacetylase 14 in a global manner by comparing the leaf lysine acetylome of a hda14 KO plant to a wild-type under low-light conditions. Proteins were extracted and digested using an adapted FASP procedure, peptides were dimethyl-labeled and lysine-acetylated peptides were antibody-enriched. Peptide identification and quantitative data analysis was carried out using MaxQuant.

2017-10-25 | PXD006651 | Pride

Polylysine interactome in human cells

Project description:Polylysine chains are synthesized by ribosomes when a stop codon preceding the polyA tail on the mRNA fails to terminate the translation. To clarify the faith of the polylysine-tagged chains, we analyzed polylysine interactome in the cytosol of human 293T cells. We found an extensive network of interactors involving key complexes of protein quality control, such as TRiC and 26S proteasome. The presence of components of the nuclear transport machinery among polylysine interactors and an enhanced nuclear import suggest a mechanism for compartmentalization of faulty proteins in the cell.

2018-04-26 | PXD008171 | Pride

Interference of RNA molecules with protein aggregation in vitro.

Project description:The analysis of RNA-protein interactomes indicates that the amount of structure in RNA molecules correlates with the capacity of binding to proteins in vitro and in vivo. To validate this observation, we used an in vitro network that mimics the composition of phase-separated RNA granules. The interference of external mRNA molecules on the protein composition of the network was analyzed by quantitative mass spectrometry.

2019-09-13 | PXD011751 | Pride

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7

Project description:This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-2 and helix S3 of VSD2 in a chimeric channel system. Sample preparation of purified chimeric channels crosslinked with the photoprobes was based on the FASP protocol. Various digestion enzymes were evaluated in order to maximise the sequence coverage of this membrane protein. Finally, MeroX was employed in order to identify the sites of crosslinking. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

2019-11-18 | PXD015037 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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