共查询到20条相似文献,搜索用时 0 毫秒
1.
Christopher Lößner Uwe Warnken Armin Pscherer Martina Schnölzer 《Analytical biochemistry》2011,(1):41530
Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. However, the accuracy of quantification is impaired by the metabolic conversion of arginine to proline resulting in additional heavy labeled proline peptide satellites. Here we reinvestigated the addition of unlabeled proline during cell cultivation under SILAC conditions considering several thousand peptides and demonstrated that the arginine-to-proline conversion is prevented independent of the cell line used. 相似文献
2.
Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days. 相似文献
3.
We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions. 相似文献
4.
Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) 总被引:1,自引:0,他引:1
Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes. 相似文献
5.
Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC) 总被引:3,自引:0,他引:3
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. 相似文献
6.
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. 相似文献
7.
Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC) 总被引:1,自引:0,他引:1
Foster LJ Rudich A Talior I Patel N Huang X Furtado LM Bilan PJ Mann M Klip A 《Journal of proteome research》2006,5(1):64-75
The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane. 相似文献
8.
Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies. 相似文献
9.
Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research 总被引:5,自引:0,他引:5
Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of (12)C- and (13)C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines. 相似文献
10.
Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d. 相似文献
11.
Identification of miRNA targets with stable isotope labeling by amino acids in cell culture 总被引:7,自引:0,他引:7
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. 相似文献
12.
Stable isotope labeling by amino acids in cell culture,SILAC, as a simple and accurate approach to expression proteomics 总被引:3,自引:0,他引:3
Ong SE Blagoev B Kratchmarova I Kristensen DB Steen H Pandey A Mann M 《Molecular & cellular proteomics : MCP》2002,1(5):376-386
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system. 相似文献
13.
Geiger T Wisniewski JR Cox J Zanivan S Kruger M Ishihama Y Mann M 《Nature protocols》2011,6(2):147-157
Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach--the use of SILAC as an internal or 'spike-in' standard--wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results. 相似文献
14.
Graumann J Hubner NC Kim JB Ko K Moser M Kumar C Cox J Schöler H Mann M 《Molecular & cellular proteomics : MCP》2008,7(4):672-683
Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks. 相似文献
15.
Chen CY Chi LM Chi HC Tsai MM Tsai CY Tseng YH Lin YH Chen WJ Huang YH Lin KH 《Molecular & cellular proteomics : MCP》2012,11(4):M111.011270
The thyroid hormone, 3, 3',5-triiodo-l-thyronine (T(3)), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T(3) are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T(3)-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TRα1 (HepG2-TRα1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T(3) target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T(3) induced PAI-1 expression in J7-TRα1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T(3)/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T(3)-treated HepG2-TRα1 cells. The T(3)-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T(3)-associated tumor progression and prognosis. 相似文献
16.
Su X Zhang L Lucas DM Davis ME Knapp AR Green-Church KB Marcucci G Parthun MR Byrd JC Freitas MA 《Analytical biochemistry》2007,363(1):22-34
This paper describes an integrated approach that couples stable isotope labeling with amino acids in cell culture to acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular medium and in medium in which lysine was substituted with deuterium-labeled lysine. Histone deacetylase (HDAC) activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture medium for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and from labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF MS. Detailed information was obtained for both the change of histone H4 acetylation specific to the N terminus and the global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. This study provides a quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli. 相似文献
17.
Romijn EP Christis C Wieffer M Gouw JW Fullaondo A van der Sluijs P Braakman I Heck AJ 《Molecular & cellular proteomics : MCP》2005,4(9):1297-1310
B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [13C6]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production. 相似文献
18.
Jehmlich N Kopinke FD Lenhard S Vogt C Herbst FA Seifert J Lissner U Völker U Schmidt F von Bergen M 《Proteomics》2012,12(1):37-42
We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions. 相似文献
19.
Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles. 相似文献
20.
Oeljeklaus S Reinartz BS Wolf J Wiese S Tonillo J Podwojski K Kuhlmann K Stephan C Meyer HE Schliebs W Brocard C Erdmann R Warscheid B 《Journal of proteome research》2012,11(4):2567-2580
The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer. 相似文献