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Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics.

ABSTRACT: Comparison of SILAC and reductive dimethyl labeling; also applied both methods to affinity enrichment experiments with and without fractionation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Shao-En Ong

PROVIDER: MSV000082868 | MassIVE | Wed Aug 29 18:11:00 BST 2018

REPOSITORIES: MassIVE

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Publications

Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics.

Lau Ho-Tak HT Suh Hyong Won HW Golkowski Martin M Ong Shao-En SE

Journal of proteome research 20140812 9

Stable isotope labeling is widely used to encode and quantify proteins in mass-spectrometry-based proteomics. We compared metabolic labeling with stable isotope labeling by amino acids in cell culture (SILAC) and chemical labeling by stable isotope dimethyl labeling and find that they have comparable accuracy and quantitative dynamic range in unfractionated proteome analyses and affinity pull-down experiments. Analyzing SILAC- and dimethyl-labeled samples together in single liquid chromatography ...[more]

PMID: 25077673

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Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics.

Project description:Stable isotope labeling is widely used to encode and quantify proteins in mass-spectrometry-based proteomics. We compared metabolic labeling with stable isotope labeling by amino acids in cell culture (SILAC) and chemical labeling by stable isotope dimethyl labeling and find that they have comparable accuracy and quantitative dynamic range in unfractionated proteome analyses and affinity pull-down experiments. Analyzing SILAC- and dimethyl-labeled samples together in single liquid chromatography-mass spectrometric analyses minimizes differences under analytical conditions, allowing comparisons of quantitative errors introduced during sample processing. We find that SILAC is more reproducible than dimethyl labeling. Because proteins from metabolically labeled populations can be combined before proteolytic digestion, SILAC is particularly suited to studies with extensive sample processing, such as fractionation and enrichment of peptides with post-translational modifications. We compared both methods in pull-down experiments using a kinase inhibitor, dasatinib, and tagged GRB2-SH2 protein as affinity baits. We describe a StageTip dimethyl-labeling protocol that we applied to in-solution and in-gel protein digests. Comparing the impact of post-digest isotopic labeling on quantitative accuracy, we demonstrate how specific experimental designs can benefit most from metabolic labeling approaches like SILAC and situations where chemical labeling by stable isotope-dimethyl labeling can be a practical alternative.

| S-EPMC4156256 | 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

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.

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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

Large scale qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

Project description:Triplex stable isotope dimethyl labeling of phosphotyrosine peptides after EGF stimulation

2010-07-09 | PRD000123 | Pride

Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).

Project description:Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced reliability in expression ratios.

| S-EPMC3394155 | biostudies-literature

Stable Isotope Labeling using Amino acids in Cell culture (SILAC) (local data base)

Project description:Stable Isotope Labeling using Amino acids in Cell culture (SILAC) of CT26 control and IF1-ablated cells

2023-10-24 | PXD038109 | Pride

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing.

Project description:Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

| S-EPMC5141271 | biostudies-literature

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

Project description:Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

| S-EPMC5341006 | biostudies-literature

Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC).

Project description:Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.

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