A multiplexed proteomics approach to differentiate neurite outgrowth patterns

Liu, Tong; D’Mello, Veera; Deng, Longwen; Hu, Jun; Ricardo, Michael; Pan, Sanqiang; Lü, Xiaodong; Wadsworth, Scott; Siekierka, John; Birge, Raymond B.; Li, Hong

doi:10.1016/j.jneumeth.2006.05.010

Cited by 15 publications

(14 citation statements)

References 23 publications

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“…We performed three independent iTRAQ experiments with different sets of mice and duplicated each sample for LC/MS/MS analysis. For iTRAQ labeling, trypsin-digested peptides were purified and desalted using an Oasis HLB extraction kit (22, 23). Concentrated peptides were reconstituted in iTRAQ reagents for 2-plex reactions (114 = ENT1 +/+ mice; 116 = ENT1 −/− mice).…”

Section: Methodsmentioning

confidence: 99%

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

Nam

Lee

Zhu

et al. 2011

Biological Psychiatry

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Background Mice lacking type 1 equilibrative nucleoside transporter (ENT1−/−) exhibit increased ethanol-preferring behavior compared to wild-type littermates. This phenotype of ENT1−/− mice appears to be correlated with increased glutamate levels in the nucleus accumbens (NAc). However, little is known about the downstream consequences of increased glutamate signaling in the NAc. Methods To investigate the significance of the deletion of ENT1 and its effect on glutamate signaling in the NAc, we employed microdialysis and iTRAQ proteomics. We validated altered proteins using Western blot analysis. We then examined the pharmacological effects of the inhibition of the N-Methyl-D-Aspartate (NMDA) glutamate receptor and protein kinase Cγ (PKCγ) on alcohol drinking in wild-type mice. In addition, we investigated in vivo cAMP response element binding (CREB) activity using CRE-lacZ mice in an ENT1−/− background. Results We identified that NMDA glutamate receptor-mediated down-regulation of intracellular PKCγ-neurogranin (Ng)-Ca2+-calmodulin dependent protein kinase type II (CaMKII) signaling is correlated with reduced CREB activity in ENT1−/− mice. Inhibition of PKCγ promotes ethanol drinking in wild-type mice to levels similar to those of ENT1−/− mice. In contrast, an NMDA glutamate receptor antagonist reduces ethanol drinking of ENT1−/− mice. Conclusion These findings demonstrate that the genetic deletion or pharmacological inhibition of ENT1 regulates NMDA glutamate receptor-mediated signaling in the NAc which provides a molecular basis that underlies the ethanol-preferring behavior of ENT1−/− mice.

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Section: Methodsmentioning

confidence: 99%

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

Nam

Lee

Zhu

et al. 2011

Biological Psychiatry

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“…This method has been effectively used for the identification of novel proteomic changes in many neurological disease models, including changes of protein expression 17 , PTM 18 , and proteolysis 19 in EAE spinal cords and other animal models of multiple sclerosis 20 . It is also an effective method for studying basic neurobiological systems, including peripheral neuroregeneration 21 , oligodendrocyte differentiation 22 and the identification of potential targets for cocaine addiction treatment 23 . Because the iTRAQ method analyzes peptides instead of intact proteins, this method is effective for the quantification of peptides that can be extracted from FFPE tissue blocks or slides.…”

Section: Introductionmentioning

confidence: 99%

Proteomic Identification of Immunoproteasome Accumulation in Formalin-Fixed Rodent Spinal Cords with Experimental Autoimmune Encephalomyelitis

Jain

Liu

et al. 2012

J. Proteome Res.

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Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the “shotgun” quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method has made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ~50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

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“…Thirty-five fractions were collected. Following PepClean C 18 spin column desalting (Pierce, Rockford, IL, USA), each peptide fraction from SCX was further separated on a reversed-phase C 18 capillary column (0.75 × 150 mm, 3 μm, 100 Å, Dionex, Sunnyvale, CA, USA) on an Ultimate LC system coupled with a Probot spotting device (Dionex) as described previously [36]. The peptides eluted from the reversed-phase LC were mixed with a MALDI matrix (5 mg/mL α-cyano-4-hydroxycinnamic acid in 60% ACN, 0.1% TFA, 5 mM ammonium monobasicphosphate, 50 fmol/μL each of glu-fibrinogen peptide ( m / z 1570.677), and adrenocorticotrophin hormone fragments 18–39 ( m / z 2465.199) and were spotted onto the stainless steel MALDI plates for MS/MS analysis.…”

Section: Methodsmentioning

confidence: 99%

Master redox regulator Trx1 upregulates SMYD1 & modulates lysine methylation

Liu

Jain

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Thioredoxin 1 (Trx1) is a antioxidant protein that regulates protein disulfide bond reduction, transnitrosylation, denitrosylation and other redox post-translational modifications. In order to better understand how Trx1 modulates downstream protective cellular signaling events following cardiac ischemia, we conducted an expression proteomics study of left ventricles (LVs) after thoracic aortic constriction stress treatment of transgenic mice with cardiac-specific over-expression of Trx1, an animal model that has been proven to withstand more stress than its non-transgenic littermates. Although previous redox post-translational modifications proteomics studies found that several cellular protein networks are regulated by Trx1-mediated disulfide reduction and transnitrosylation, we found that Trx1 regulates the expression of a limited number of proteins. Among the proteins found to be upregulated in this study was SET and MYND domain-containing protein 1 (SMYD1), a lysine methyltransferase highly expressed in cardiac and other muscle tissues and an important regulator of cardiac development. The observation of SMYD1 induction by Trx1 following thoracic aortic constriction stress is consistent with the retrograde fetal gene cardiac protection hypothesis. The results presented here suggest for the first time that, in addition to being a master redox regulator of protein disulfide bonds and nitrosation, Trx1 may also modulate lysine methylation, a non-redox post-translational modification, via the regulation of SMYD1 expression. Such crosstalk between redox signaling and a non-redox PTM regulation may provide novel insights into the functions of Trx1 that are independent from its immediate function as a protein reductase.

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A multiplexed proteomics approach to differentiate neurite outgrowth patterns

Cited by 15 publications

References 23 publications

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking Through Accumbal N-Methyl-D-Aspartate Receptor Signaling

Proteomic Identification of Immunoproteasome Accumulation in Formalin-Fixed Rodent Spinal Cords with Experimental Autoimmune Encephalomyelitis

Master redox regulator Trx1 upregulates SMYD1 & modulates lysine methylation

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