Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

ABSTRACT: This project contains intact protein MS and PRM data for several central metabolic enzymes in E.coli. The enzymes are both wild-type and mutant for several

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Peter Doubleday

LAB HEAD: Uwe Sauer

PROVIDER: PXD027243 | Pride | 2021-08-20

REPOSITORIES: pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20201124_pfd_MAbPac_Gnd_S304A_01.raw	Raw
	20201124_pfd_MAbPac_Gnd_S304A_PRM_01.raw	Raw
	20201124_pfd_MAbPac_Gnd_S304A_PRM_02.raw	Raw
	20201124_pfd_MAbPac_Gnd_S304E_01.raw	Raw
	20201124_pfd_MAbPac_Gnd_S304E_PRM_01.raw	Raw

Items per page:

1 - 5 of 77

Similar Datasets

Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli

Project description:Here we investigate the role of acetyl phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated from central metabolsim, only GapA and GpmA were acetylated at high stoichiometry.

2022-12-16 | PXD035653 | Pride

Development of a top-down MS assay for specific identification of human periostin isoforms

Project description:Periostin is a matricellular protein encoded by the POSTN gene that is alternatively spliced to produce ten different isoforms of periostin with a molecular weight ranging from 78 to 91 kDa. Periostin is known to promote fibrillogenesis, organize the extracellular matrix, and bind integrin-receptors to induce cell signaling. Periostin is a key component of the wound healing process but is also known to participate in the pathogenesis of different diseases including atopic dermatitis, asthma, and cancer. In both health and disease, the functions of the different periostin isoforms are largely unknown. The ability to precisely determine the isoform profile of a given human sample is fundamental for characterizing their functional significance. Identification of periostin isoforms is most often carried out at the transcriptional level using RT-PCR based approaches. Though, it must be recognized that periostin exerts it functions at the translational level and is an extracellular protein making it impossible to derive transcriptional information for e.g. plasma periostin that is spatially disconnected from the cell it was expressed by. Consequently, monitoring periostin isoforms at the protein level is highly advantageous. In the publication connected to this dataset we present a fully developed top down mass spectrometry assay for identification and quantification of periostin splice isoforms from any human sample at the protein level.

2024-06-11 | PXD045560 | Pride

Online electrochemical reduction of both inter- and intramolecular disulfide bridges in immunoglobulins

Project description:Cysteine disulfide bridges can be reduced by chemical methods, like an incubation with DTT. However, reduction with an electrochemical method has also been demonstrated. Electrochemical reduction can be implemented online, after LC separation and before mass spectrometry. For the study of antibody molecules, reduction of the intermolecular disulfide bridges between heavy and light chains has been demonstrated in literature. However, the reduction of intramolecular disulfide bridges has remained elusive. Here, we demonstrate the online electrochemical reduction of a therapeutic monoclonal antibody. The complete reduction of these molecules will facilitate the study of recombinant or natural antibodies by MS and MS/MS.

2022-05-27 | PXD021649 | Pride

TDFragMapper: Evaluating experimental parameters for confident intact protein characterization in top-down proteomics

Project description:New software-tool allowing an easy visualization of fragment ions and thus a rapid evaluation of key experimental parameters on the sequence coverage obtained for the MS/MS analysis of intact proteins. Our tool can deal with multiple fragmentation methods. We demonstrate that TDFragMapper can rapidly highlight the experimental fragmentation parameters that are critical to the characterization of intact proteins of various size using top-down proteomics.

2021-12-30 | PXD024643 | Pride

A multi-parameter optimization in middle-down analysis of monoclonal antibodies by LC-MS/MS

Project description:In antibody-based drug research, regulatory agencies request a complete characterization of antibody proteoforms covering both the amino acid sequence and all post-translational modifications. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies, but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states and the combination of replicates.

2023-03-15 | PXD038619 | Pride

Top-down analysis of purified IL-38

Project description:Interleukin (IL)-1 family cytokines are essential for host defense at epithelial barriers. The IL-1 family member IL-33 was recently linked to stress granules (SGs). Formation of SGs and other biomolecular condensates is promoted by proteins containing low complexity regions (LCRs). Computational analysis predicted LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR including two amyloid cores. IL-38 localized to intracellular condensates in keratinocytes exposed to oxidative stress (OS) and formed OS-induced amyloid aggregates in cells and in vitro. Top-down proteomic analysis was performed on purified recombinant IL-38 protein to characterize the presence of possible disulfide bonds.

2024-11-03 | PXD052918 | Pride

Benchmarking multiple fragmentation methods on an Orbitrap Fusion for top-down phospho-proteoform characterization

Project description:Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms.

2015-07-16 | PXD001845 | Pride

Characterization of human sperm protamine proteoforms through a combination of top-down and bottom-up mass spectrometry approaches

Project description:Protamine 1 (P1) and protamine 2 family (P2) are the most abundant nuclear basic spermspecific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3 and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies are not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile including P1 and P2 members and their post-translational modifications in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase K-digestion based bottom-up MS approach. By top-down MS approach, both described and new truncated P1 and pre-P2 proteoforms were identified. Likewise, intact P1, pre-P2, and P2 mature forms and their phosphorylation pattern were detected, as well as a +61 Da modification in different proteoforms. Additionally, the bottom-up MS approach revealed phosphorylated residues for pre-P2 and the new P2 isoform 2, which is not annotated at UniProtKB database. Implementing these strategies in comparative studies of different infertile phenotypes would permit to determine alterations in the protamine proteoforms pattern and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

2019-11-11 | PXD014618 | Pride

Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top-down and middle-down mass spectrometry

Project description:We propose here a workflow based on an automated immunocapture followed by top-down and middle-down LC-MS/MS approaches to characterize mAb proteoforms spiked in mouse plasma. We demonstrate the applicability of our workflow on a large concentration range using pembrolizumab as a model. We also compare the performance of two state-of-the-art Orbitrap platforms (Tribrid Eclipse and Exploris 480) for these studies. The added value of our workflow for an accurate and sensitive characterization of mAb proteoforms in mouse plasma is highlighted.

2024-08-27 | PXD040896 | Pride

Mass-Spectrometric analysis of SdeA Ubiquitin modification and Ubiquitination

Project description:Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C-terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Yet, the molecular mechanism of this ubiquitination reaction and its relevance for cellular processes remained elusive. Here, we report the identification of a phosphodiesterase (PDE) domain in SdeA that efficiently catalyses arginine phosphoribosylation of ubiquitin via an ADP-ribose intermediate . The PDE domain also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade thereby diminishing numerous cellular processes including mitophagy, TNF signaling and proteasomal degradation. We propose that phosphoribosylation of ubiquitin is a potent modulator of ubiquitin functions in mammalian cells.

2016-12-07 | PXD005240 | 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