Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Scalable multiplex co-fractionation/mass spectrometry platform for accelerated protein interactome discovery.

ABSTRACT: Co-fractionation/mass spectrometry (CF/MS) enables the mapping of endogenous macromolecular networks on a proteome scale, but current methods are experimentally laborious, resource intensive and afford lesser quantitative accuracy. Here, we present a technically efficient, cost-effective and reproducible multiplex CF/MS (mCF/MS) platform for measuring and comparing, simultaneously, multi-protein assemblies across different experimental samples at a rate that is up to an order of magnitude faster than previous approaches. We apply mCF/MS to map the protein interaction landscape of non-transformed mammary epithelia versus breast cancer cells in parallel, revealing large-scale differences in protein-protein interactions and the relative abundance of associated macromolecules connected with cancer-related pathways and altered cellular processes. The integration of multiplexing capability within an optimized workflow renders mCF/MS as a powerful tool for systematically exploring physical interaction networks in a comparative manner.

SUBMITTER: Havugimana PC

PROVIDER: S-EPMC9279285 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Co-fractionation/mass spectrometry to identify protein complexes.

Project description:Co-fractionation/mass spectrometry (CF/MS) is a flexible and powerful method to detect physical associations of proteins. CF/MS can be applied to any tissue or organism without the need for protein-specific antibodies or epitope tags. Here, we outline two alternate protocols for MS preparation of samples (containing low or high salt) and a computational pipeline (cfmsflow) that together allow the successful application of this approach. These protocols are based on CF/MS of over 16 diverse organisms including plants and animals. For complete details on the use and execution of this protocol, please refer to McWhite et al. (2020).

| S-EPMC7960544 | biostudies-literature

Mass Spectrometry-Based Screening Platform Reveals Orco Interactome in Drosophila melanogaster.

Project description:Animals use their odorant receptors to receive chemical information from the environment. Insect odorant receptors differ from the G protein-coupled odorant receptors in vertebrates and nematodes, and very little is known about their protein-protein interactions. Here, we introduce a mass spectrometric platform designed for the large-scale analysis of insect odorant receptor protein-protein interactions. Using this platform, we obtained the first Orco interactome from Drosophila melanogaster. From a total of 1,186 identified proteins, we narrowed the interaction candidates to 226, of which only two-thirds have been named. These candidates include the known olfactory proteins Or92a and Obp51a. Around 90% of the proteins having published names likely function inside the cell, and nearly half of these intracellular proteins are associated with the endomembrane system. In a basic loss-of-function electrophysiological screen, we found that the disruption of eight (i.e., Rab5, CG32795, Mpcp, Tom70, Vir-1, CG30427, Eaat1, and CG2781) of 28 randomly selected candidates affects olfactory responses in vivo. Thus, because this Orco interactome includes physiologically meaningful candidates, we anticipate that our platform will help guide further research on the molecular mechanisms of the insect odorant receptor family.

| S-EPMC5824025 | biostudies-literature

A scalable multiplexed platform to accelerate protein interactome discovery

Project description:Chromatographic fractionation-mass spectrometry (CF-MS) allows for mapping native protein-protein interactions and macromolecular complexes on a proteome-scale but is tedious and resource intensive for comparative analysis. Here, we present a more parsimonious and reproducible multiplexing CF-MS platform for measuring multi-protein assemblies across different experimental samples simultaneously with an order-of-magnitude faster than previous approaches. We used this innovative strategy to elucidate protein networks in control and breast cancer cells models in parallel within two weeks of total MS instrument time.

2022-08-03 | PXD027704 | Pride

Mapping protein states and interactions across the tree of life with co-fractionation mass spectrometry.

Project description:We present CFdb, a harmonized resource of interaction proteomics data from 411 co-fractionation mass spectrometry (CF-MS) datasets spanning 21,703 fractions. Meta-analysis of this resource charts protein abundance, phosphorylation, and interactions throughout the tree of life, including a reference map of the human interactome. We show how large-scale CF-MS data can enhance analyses of individual CF-MS datasets, and exemplify this strategy by mapping the honey bee interactome.

| S-EPMC10724252 | biostudies-literature

Differential Peptide Subtraction (DPS): mass spectrometry for human cancer virus discovery

Project description:The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host-derived molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. To trace the non-human dMS identified peptides back to its genetic origin by next generation sequencing (NGS) cDNA libraries were generated using degenerate oligos corresponding to the identified peptides. In a pilot study DPS identified both viral and human biomarkers. Based on the identified peptides that are differentially expressed in the virus positive tumor sample, degenerate oligos were designed. Degenerate oligos or LNA modified degenerate oligos or a modified oligo(dT) SMART primer were used to facilitate reverse transcription from viral or viral and host RNA. NGS analysis revealed seven times more MCV reads in degenerate oligo primed RNAseq samples compared to polyA-based sequencing reads.

2020-10-20 | GSE157610 | GEO

Automated sample preparation platform for mass spectrometry-based plasma proteomics and biomarker discovery.

Project description:The identification of novel biomarkers from human plasma remains a critical need in order to develop and monitor drug therapies for nearly all disease areas. The discovery of novel plasma biomarkers is, however, significantly hampered by the complexity and dynamic range of proteins within plasma, as well as the inherent variability in composition from patient to patient. In addition, it is widely accepted that most soluble plasma biomarkers for diseases such as cancer will be represented by tissue leakage products, circulating in plasma at low levels. It is therefore necessary to find approaches with the prerequisite level of sensitivity in such a complex biological matrix. Strategies for fractionating the plasma proteome have been suggested, but improvements in sensitivity are often negated by the resultant process variability. Here we describe an approach using multidimensional chromatography and on-line protein derivatization, which allows for higher sensitivity, whilst minimizing the process variability. In order to evaluate this automated process fully, we demonstrate three levels of processing and compare sensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of the human plasma proteome is possible down to the low ng/mL or even high pg/mL level with a high degree of technical reproducibility.

| S-EPMC4009759 | biostudies-literature

A co-fractionation mass spectrometry-based prediction of protein complex assemblies in the developing rice aleurone-subaleurone.

Project description:Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and system-level control. This is of great biological importance and practical significance in heterotrophic rice (Oryza sativa) endosperm and aleurone-subaleurone tissues, which are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this study was to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable large-scale multimerization state measurements and protein complex predictions. The predicted complexes had predicted functions across diverse functional categories, including novel heteromeric RNA binding protein complexes that may influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about system-level posttranslational control during the early stages of rice seed development.

| S-EPMC8462808 | biostudies-literature

An integrated mass spectrometry platform enables picomole-scale real-time electrosynthetic reaction screening and discovery.

Project description:The identification of new electrosynthetic pathways enables environmentally friendly synthetic applications. However, the development of miniaturized screening procedures/platforms to expedite the discovery of electrooxidation reactions remains challenging. Herein, we developed an integrated system that serves as a reactor and ion source in a single experimental step using only picomole-scale reactants to monitor electrooxidation in real-time. This reaction screening platform utilizes the intrinsic electrochemical capabilities of nano-electrospray ionization mass spectrometry. We validated the feasibility of this method by reproducing three known electrochemical reactions. We also discovered two new electroorganic reaction pathways: (i) C-N dehydrodimerization of 8-methyl-1,2,3,4-tetrahydroquinoline to construct a novel quinoline skeleton, and (ii) TEMPO-mediated accelerated electrooxidative dehydrogenation of tetrahydroisoquinolines. Moreover, the radical cations and key intermediates captured by this screening platform provided direct evidence for the mechanism of these novel electrochemical reactions.

| S-EPMC6050606 | biostudies-literature

Comprehensive mass spectrometry based biomarker discovery and validation platform as applied to diabetic kidney disease.

Project description:A protein biomarker discovery workflow was applied to plasma samples from patients at different stages of diabetic kidney disease. The proteomics platform produced a panel of significant plasma biomarkers that were statistically scrutinised against the current gold standard tests on an analysis of 572 patients. Five proteins were significantly associated with diabetic kidney disease defined by albuminuria, renal impairment (eGFR) and chronic kidney disease staging (CKD Stage ≥1, ROC curve of 0.77). The results prove the suitability and efficacy of the process used, and introduce a biomarker panel with the potential to improve diagnosis of diabetic kidney disease.

| S-EPMC5988498 | biostudies-literature

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics.

Project description:Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopus laevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.

| S-EPMC9024316 | biostudies-literature

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