Peptide Mass Mapping Constrained with Stable Isotope-Tagged Peptides for Identification of Protein Mixtures

Hunter, Thomas C.; Li, Yang; Zhu, Haining; Majidi, Vahid; Bradbury, E. Morton; Chen, Xian

doi:10.1021/ac0103322

Cited by 45 publications

(52 citation statements)

References 45 publications

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“…WT-SOD1 and NSC34 G93A-SOD1 cells was carried out as previously described (54). Briefly, the mitochondrial pellet was resuspended and homogenized in rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer 3-10) (ϳ100 to 150 g of protein in 50 l of buffer) and centrifuged at 10,000 ϫ g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…After 2D gel electrophoresis of the mitochondrial proteins, 480 protein spots of greatest density were excised from the gel. All gel bands or pieces were washed and digested with trypsin as previously described (54,55). The resulting peptides were extracted using 50 l of 0.02% heptafluorobutylic acid (HFBA) and 50 l of 0.02% HFBA/50% ACN and concentrated to 10 l with a Speed Vac.…”

Section: Methodsmentioning

confidence: 99%

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Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Fukada

Zhang

Vien

et al. 2004

Molecular & Cellular Proteomics

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115

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Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. Amyotrophic lateral sclerosis (ALS)1 is a fatal neurodegenerative disease characterized by progressive motor neuron death. Approximately 10% of ALS patients are familial cases (fALS), and mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) were linked with a subset of fALS (1, 2). To date, more than 90 mutations in SOD1 are known to be responsible for ϳ25% of fALS (3), most of which are point mutations that are scattered throughout the primary sequence and structure of the protein. There has been intensive research focusing on the etiology of SOD1 mutant-mediated fALS (see reviews in . It has been demonstrated that SOD1-null mice did not develop the disease (9). In addition, transgenic mice expressing the ALS-associated mutants G93A-SOD1 (10, 11), G37R-SOD1 (12), and G85R-SOD1 (13, 14) as well as transgenic rats expressing G93A-SOD1 (15, 16) and H46R-SOD1 (16) developed progressive motor neuron disease despite normal or elevated SOD1 activity. Therefore, it is believed that the ALS-linked mutants of SOD1 have acquired unknown toxic properties that eventually lead to the disease. However, ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Fukada

Zhang

Vien

et al. 2004

Molecular & Cellular Proteomics

Self Cite

115

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“…Matching of two-dimensional images (three from LNCaP cell and three from LNCaP-ThRII) was done automatically, followed by manual matching. Protein spots were excised and in-gel digestion was done as previously described (24,25). The resulting peptides were extracted with 0.02% heptafluorobutylic acid and 0.02% heptafluorobutylic acid/50% acetonitrile and subsequently subjected to liquid chromatography-tandem mass spectrometry (MS/MS) analysis followed by scanning with Mascot.…”

Section: Analysis Of Two-dimensional Electrophoresis Imagesmentioning

confidence: 99%

Prohibitin and Cofilin Are Intracellular Effectors of Transforming Growth Factor β Signaling in Human Prostate Cancer Cells

Zhu¹,

Fukada

Zhu

et al. 2006

Cancer Research

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A proteomic analysis was pursued to identify new signaling effectors of transforming growth factor B1 (TGF-B1) that serve as potential intracellular effectors of its apoptotic action in human prostate cancer cells. The androgen-sensitive and TGF-B-responsive human prostate cancer cells, LNCaP TBRII, were used as in vitro model. In response to TGF-B, significant posttranslational changes in two proteins temporally preceded apoptotic cell death. TGF-B mediated the nuclear export of prohibitin, a protein involved in androgen-regulated prostate growth, to the cytosol in the LNCaP TBRII cells. Cofilin, a protein involved in actin depolymerization, cell motility, and apoptosis, was found to undergo mitochondrial translocation in response to TGF-B before cytochrome c release. Loss-offunction approaches (small interfering RNA) to silence prohibitin expression revealed a modest decrease in the apoptotic response to TGF-B and a significant suppression in TGF-B-induced cell migration. Silencing Smad4 showed that the cellular localization changes associated with prohibitin and cofilin action in response to TGF-B are independent of

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“…An abundance ratio between the labeled and unlabeled peptides can then be calculated from the respective ion chromatograms Gruhler et al 2005;Everley et al 2006;Pan et al 2006). Metabolic labeling is commonly employed to enrich a protein sample with stable isotopes (Goodlett et al 2001;Hunter et al 2001;Ong et al 2002;Washburn et al 2002;Zhu et al 2002), and then, alterations in protein expression induced by a N ratios between the two experiments reveals protein expression altered by osmotic shock . Besides yeast, metabolic labeling of cultured mammalian cells using one or two stable isotope-labeled amino acids strategy is commonly employed to generate high-throughput quantitative data (Zhu et al 2002;Blagoev et al 2003).…”

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confidence: 99%

Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

McClatchy

Liao²,

Park³

et al. 2007

Genome Res.

100

110

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Large-scale proteomic analysis of the mammalian brain has been successfully performed with mass spectrometry techniques, such as Multidimensional Protein Identification Technology (MudPIT), to identify hundreds to thousands of proteins. Strategies to efficiently quantify protein expression levels in the brain in a large-scale fashion, however, are lacking. Here, we demonstrate a novel quantification strategy for brain proteomics called SILAM (Stable Isotope Labeling in Mammals). We utilized a 15 N metabolically labeled rat brain as an internal standard to perform quantitative MudPIT analysis on the synaptosomal fraction of the cerebellum during post-natal development. We quantified the protein expression level of 1138 proteins in four developmental time points, and 196 protein alterations were determined to be statistically significant. Over 50% of the developmental changes observed have been previously reported using other protein quantification techniques, and we also identified proteins as potential novel regulators of neurodevelopment. We report the first large-scale proteomic analysis of synaptic development in the cerebellum, and we demonstrate a useful quantitative strategy for studying animal models of neurological disease.

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Peptide Mass Mapping Constrained with Stable Isotope-Tagged Peptides for Identification of Protein Mixtures

Cited by 45 publications

References 45 publications

Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Prohibitin and Cofilin Are Intracellular Effectors of Transforming Growth Factor β Signaling in Human Prostate Cancer Cells

Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

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