Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits.

ABSTRACT: Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[¹³C₆] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.

SUBMITTER: Schober FA

PROVIDER: S-EPMC8050774 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Stable-isotope labeling with amino acids in nematodes.

Project description:We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine- and heavy arginine-labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.

| S-EPMC3184259 | biostudies-literature

Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.

Project description:Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.

| S-EPMC3443408 | biostudies-literature

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.

Project description:miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3'-untranslated region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins.

| S-EPMC1636363 | biostudies-literature

Stable isotope labeling with amino acids in mouse xenografts.

Project description:This dataset corresponds to a SILAC analysis in mouse xenografts and in cell culture. We analysed signalling in tumors by means of a method based on stable isotope labeling with amino acids of xenograft tumors by the addition of [(13)C(6)]-lysine into mouse food, and also using a typical SILAC analysis in cell culture. This dataset includes 2 groups of analysis: one, with maxquant file tagged SILAX, corresponding to the xenograft analysis, the other one to SILAC analysis in cell culture. Data were acquired on an LTQ-Orbitrap XL mass spectrometer coupled with an Ultimate 3000 HPLC. Analysis was performed using MaxQuant software (version 1.1.1.36). All MS/MS spectra were searched using Andromeda against a database consisting of a combination of Homo sapiens and Mus musculus CPS databases and 250 classical contaminants. Search was performed allowing the following variable modifications: Oxidation (M) and Phospho (STY). FDR was set at 0.01 for peptides and proteins, and the minimal peptide length was six. Quantification was also performed using standard parameters; we considered only proteins with at least two identified/quantified peptides.

2013-01-17 | PXD000044 | Pride

Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis.

Project description:Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker's yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker's yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).

| S-EPMC6300314 | biostudies-literature

Intercellular transfer of proteins as identified by stable isotope labeling of amino acids in cell culture.

Project description:We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems. We find that irradiation to murine lung and lung-derived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines. The functional classification of transferred proteins was broad and included transcription factors, mediators of basic cellular processes and components of the nucleosome remodeling and deacetylase complex, including metastasis associated protein 3 and retinoblastoma-binding protein 7. In further analysis we find that retinoblastoma-binding protein 7 is a transcriptional activator of E-cadherin and that its intercellular transfer leads to decreased gene expression of downstream targets such as N-cadherin and vimentin. SILAC-generated data sets offer a valuable tool to identify and validate potential paracrine networks that may impact relevant biologic processes associated with phenotypic and genotypic signatures of health and disease.

| S-EPMC2825424 | biostudies-literature

Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

Project description:Background and objectiveStable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit.MethodsWe introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum.ResultsOur simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data.ConclusionsNo matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes.

| S-EPMC5260509 | biostudies-literature

Stable Isotope Labeling using Amino acids in Cell culture (SILAC) (mus.musculus)

Project description:Targeted SILAC determination of the rate of synthesis of proteins involved in mitochondrial structure and ETC activity in CT26 control and IF1-ablated cell lines

2023-10-24 | PXD038108 | Pride

Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).

Project description:Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios.

| S-EPMC2542903 | biostudies-literature

IsoQuant: a software tool for stable isotope labeling by amino acids in cell culture-based mass spectrometry quantitation.

Project description:Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics data sets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high-throughput data sets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to noncommercial users at http://www.proteomeumb.org/MZw.html .

| S-EPMC3583527 | 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