Identification of Differentially Expressed Proteins in Experimental Autoimmune Encephalomyelitis (EAE) by Proteomic Analysis of the Spinal Cord

Liu, Tong; Donahue, Katie; Hu, Jun; Kurnellas, Michael; Grant, Jennifer E.; Li, Hong; Elkabes, Stella

doi:10.1021/pr070012k

Cited by 58 publications

(92 citation statements)

References 83 publications

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“…Peptide identification was performed using the following parameters: Cys alkylation: MMT(C), ID focus: biological modification, Digestion: trypsin, Database: zebrafish, Search effort: thorough ID, unused protein score >1.3 (>95% confidence). Protein Pilot 4.0 software (AB Sciex, Foster City, CA, USA) was used for relative quantification of proteins, and the expression level was considered statistically significant if P < 0.05 and exhibited a fold change >1.4 for upregulation and <0.7 for downregulation as described (Liu et al, 2007). iTRAQ 8-plex was used in the present study, which can technically only measure 8 samples in one run, therefore, there were two biological replicates for control group and three biological replicates for treatment groups in this study and each biological replicate included 80 larvae.…”

Section: Quantitative Proteomic Analysismentioning

confidence: 99%

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

Han

Wang

et al. 2014

Aquatic Toxicology

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Section: Quantitative Proteomic Analysismentioning

confidence: 99%

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

Han

Wang

et al. 2014

Aquatic Toxicology

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“…values no less than 95% were used for protein identification and quantitation. Protein expression ratios (Tg-Trx1/control) were calculated as described previously (25,26). Briefly the iTRAQ reporter ion cluster areas were extracted using the GPS Explorer software (ABI), and only ion counts greater than 5,000 were used for quantification analysis.…”

Section: Ms and Database Searchmentioning

confidence: 99%

Elucidation of Thioredoxin Target Protein Networks in Mouse

Liu

et al. 2009

Molecular & Cellular Proteomics

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Thioredoxin 1 (Trx1) is a key redox modulator that is functionally conserved across a wide range of species, including plants, bacteria, and mammals. Using a conserved CXXC motif, Trx1 catalyzes the reduction of cysteine disulfides and S-nitrosothiols. In contrast to small molecular reductants such as glutathione and cysteine that can reduce a wide range of oxidized proteins, Trx1 reduces only selected proteins via specific protein-protein interaction. Trx1 has been shown to regulate numerous signal transduction pathways, and its dysfunctions have been implicated in several diseases, including cancer, inflammation, and neurodegenerative and cardiovascular diseases. Identification of Trx1 target proteins may help to identify novel signaling mechanisms that are important for Trx1 antistress responses. In this study, we performed an ICAT proteomics study for the identification of Trx1 target proteins from the hearts of a cardiac specific Trx1-overexpressing transgenic mouse model (Tg-Trx1). Trx1-reduced proteins were distinguished from Trx1-induced proteins by comparison of the ICAT results with those obtained using a parallel iTRAQ (isobaric tags for relative and absolute quantitation) protein expression analysis. We were able to identify 78 putative Trx1 reductive sites in 55 proteins. Interestingly we identified a few protein functional networks that had not been shown previously to be regulated by Trx1, including the creatine-phosphocreatine shuttle, the mitochondrial permeability transition pore complex, and the cardiac contractile apparatus. The results presented here suggest that in addition to a general antioxidant function, Trx1 may be involved in the coordination of a wide array of cellular functions for maintaining proper cardiac energy dynamics and facilitating muscle contraction. Molecular & Cellular Proteomics 8:1674 -1687, 2009. Thioredoxins (Trxs)1 are a class of antioxidant proteins that mediate the reduction of specific disulfide bonds and Snitrosothiols within oxidized proteins. Trx and the NADPH-dependent thioredoxin reductase (TrxR) form a protein reductive system that plays essential roles in the clearance of elevated reactive oxygen species, the repair of oxidatively modified proteins, and the restoration of cellular redox homeostasis. Two isoforms of Trx have been widely studied: Trx1 is mostly found in the cytosol and nucleus, whereas Trx2 is mitochondrion-specific. Each Trx isoform is coupled with its own TrxR systems. Trx1 has been shown to promote cell growth and proliferation and to inhibit apoptosis by modulating both caspase-dependent and -independent pathways. Trx1 has also been demonstrated to be a potent regulator of a wide variety of transcription factors and other gene expression regulators by preserving the reductive states of specific cysteines within transiently oxidized proteins such as nuclear factor B (1), hypoxia inducible factor-1␣ (2), histone deacetylase 4 (3), and glucocorticoid receptor (4). A wide range of bioanalytical techniques has been applied to identify Trx1 ta...

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“…The RSD of differential expression ratios was in the range of ϳ1%-67%. Currently, there has been growing interest in developing proteomic quantitative protocols for a variety of biological applications that involve, for example, the study of protein-protein interactions [16], the monitoring of temporal changes in perturbed signaling pathways [18], and the discovery of novel disease biomarkers in samples of biological origin [27][28][29][30]. For example, Keshamouni et al performed differential protein expression analysis of lung cancer cells undergoing epithelialmesenchymal transition using iTRAQ followed by 2DLC-MS/MS [29].…”

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confidence: 99%

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Armenta

Hoeschele

Lazar

2009

J. Am. Soc. Mass Spectrom.

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An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

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Identification of Differentially Expressed Proteins in Experimental Autoimmune Encephalomyelitis (EAE) by Proteomic Analysis of the Spinal Cord

Cited by 58 publications

References 83 publications

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

Elucidation of Thioredoxin Target Protein Networks in Mouse

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

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