Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS

Unwin, Richard D.; Griffiths, John R.; Whetton, Anthony D.

doi:10.1038/nprot.2010.123

Cited by 224 publications

(179 citation statements)

References 20 publications

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“…For subsequent analysis by low-pH reversed-phase LC-tandem MS analysis, dried fractions were resuspended in 15 mL 3% v/v acetonitrile and 0.1% v/v trifluoroacetic acid, with 5 mL analyzed by low-pH reversed-phase chromatography using a nanoACQUITY LC system (Waters) online to a QSTAR Elite mass spectrometer (AB Sciex) as previously described (15).…”

Section: Proteomics Sample Fractionation and Msmentioning

confidence: 99%

“…Proteomics was performed using 8-plex iTRAQ (AB Sciex) (isobaric tags for relative and absolute quantitation), a labeled method that allows simultaneous analysis of eight independent tissue samples as previously described (15) but with the following modifications. Tissue was lysed in 1 mol/L triethylammonium bicarbonate (TEAB) with 0.1% weight for volume SDS (SN 600 mL, DRG 150 mL, TG 250 mL in TissueLyser II).…”

Section: Proteomics Sample Fractionation and Msmentioning

confidence: 99%

“…To identify proteins from their peptide spectra, raw data files were analyzed with ProteinPilot 4.0 using default search settings against a rat-specific Uniprot database (15,190 proteins; release 2011_04), which was concatenated with a reversed-sequence decoy version of the same database to enable the FDR for identifications to be determined.…”

Section: Proteomic Analysismentioning

confidence: 99%

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Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental Diabetic Neuropathy

et al. 2015

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High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However, our understanding of the molecular mechanisms that cause the marked distal pathology is incomplete. We performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN. We integrated proteomics and metabolomics from the sciatic nerve (SN), the lumbar 4/5 dorsal root ganglia (DRG), and the trigeminal ganglia (TG) of streptozotocin-diabetic and healthy control rats. Even though all tissues showed a dramatic increase in glucose and polyol pathway intermediates in diabetes, a striking upregulation of mitochondrial oxidative phosphorylation and perturbation of lipid metabolism was found in the distal SN that was not present in the corresponding cell bodies of the DRG or the cranial TG. This finding suggests that the most severe molecular consequences of diabetes in the nervous system present in the SN, the region most affected by neuropathy. Such spatial metabolic dysfunction suggests a failure of energy homeostasis and/or oxidative stress, specifically in the distal axon/Schwann cell-rich SN. These data provide a detailed molecular description of the distinct compartmental effects of diabetes on the PNS that could underlie the distal-proximal distribution of pathology.Diabetic neuropathy (DN) will develop in ;30-50% of patients with diabetes. DN typically presents with sensory symptoms in a distal glove-and-stocking distribution (1,2). It is a poorly understood complication of diabetes, and currently, few treatments are available (3). Raised glucose has long been believed to instigate pathology in DN either through direct neurotoxicity or from the activation of secondary pathways (4,5). However, exactly how these pathways cause nerve conduction velocity deficits, neuropathic pain, distal axonopathy, and numbness in the extremities continues to elude us, and many clinical trials aimed at specific targets have failed due to lack of efficacy (3).Crossover exists between proposed pathogenic pathways in DN, but how these interact is unclear. This question can be approached by implementing extensive -omic technologies to measure many transcripts, proteins, or metabolites in parallel. Gene microarrays were among the first of these technologies to be used, and transcriptomic analyses have been performed on tissues such as the dorsal root ganglia (DRG) from streptozotocin (STZ)-diabetic rats compared with healthy controls (6), the sciatic nerve (SN) of db/db mice compared with those from db/+ mice (7), and sural nerve biopsy specimens from human patients whose neuropathy progressed over 1 year compared with those whose neuropathy did not (based on myelinated fiber density loss) (8). Common changes across these gene array studies highlight altered carbohydrate and lipid metabolism.Because gene transcript levels do not always correlate with protein expression due to varying transcriptional/translational

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Section: Proteomics Sample Fractionation and Msmentioning

confidence: 99%

Section: Proteomics Sample Fractionation and Msmentioning

confidence: 99%

Section: Proteomic Analysismentioning

confidence: 99%

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Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental Diabetic Neuropathy

et al. 2015

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“…Considering the low cellular concentration of ceramides, it is important to concurrently determine the concentration ratios of ceramides from different samples, such as before and after stimulation of cells. However, simultaneous analysis of ceramide levels across multiple samples has not been reported to date.Isobaric tags for relative and absolute quantitation (iTRAQ) have been developed to compare the protein expression levels in multiple samples ( 19,20 ). These reagents have identical overall mass but vary in terms of the distribution of heavy carbon, nitrogen, and oxygen isotopes within their structure.…”

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confidence: 99%

Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

Nabetani

Makino

Hullin-Matsuda

et al. 2011

Journal of Lipid Research

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This article is available online at http://www.jlr.orgCeramides are central intermediates of sphingolipid biosynthesis and degradation ( 1,2 ). In addition to their metabolic importance, ceramides play a crucial role in a variety of signaling events, including differentiation ( 3 ), senescence ( 4 ), proliferation ( 5 ), and apoptosis ( 6, 7 ). Due to their physiological importance, analysis of their generation as well as their quantifi cation has received considerable attention. Ceramides are quantitatively relatively minor cellular lipid components; thus, highly sensitive detection is required for quantifi cation. Several methods of quantifying ceramide have been reported, including gas chromatography (GC) ( 8 ), high-performance liquid chromatography (HPLC) ( 9-11 ), diacylglycerol kinase assay ( 12 ), and mass spectrometry (MS) ( 13-18 ). Recent advances in MS have made this method a promising approach to the quantitative measurement of ceramides and other lipids. Considering the low cellular concentration of ceramides, it is important to concurrently determine the concentration ratios of ceramides from different samples, such as before and after stimulation of cells. However, simultaneous analysis of ceramide levels across multiple samples has not been reported to date.Isobaric tags for relative and absolute quantitation (iTRAQ) have been developed to compare the protein expression levels in multiple samples ( 19,20 ). These reagents have identical overall mass but vary in terms of the distribution of heavy carbon, nitrogen, and oxygen isotopes within their structure. One big advantage of iTRAQ is enhanced sensitivity because the intensity of the peaks Abstract Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantifi cation. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. 31 March 2011. Published, JLR Papers in Press, April 11, 2011 DOI 10.1194 Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ) Abbreviations: MDCK, Madin Darby canine kidney; MRM, multiple reaction monitoring; PPS, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl] propane-1-sulfonate; SCDase, sphingolipid ceramide N -deacylase; SMase, sphingomyelinase; iTRAQ, isobaric tags for relative and absolute quantitation. Manuscript received 7 February 2011 and in revised form

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“…The following filters were used in this study, peptide FDR ≤ 0.01 and each protein with at least 2 unique peptides. Expression changes of the identified peptides in the regenerating head fragments were calculated in comparison with the control based on the iTRAQ reporter ion intensities (Unwin et al, 2010). Only unique peptides were utilized to determine protein quantification.…”

Section: Discussionmentioning

confidence: 99%

iTRAQ-based proteomic analysis of adaptive response in the regenerating limb of the Cynops orientalis newt

Geng¹,

Guo²,

Zang³

et al. 2015

Int. J. Dev. Biol.

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The newt has the powerful capacity to regenerate lost limbs following amputation, and represents an excellent model organism to study regenerative processes. However, the molecular basis of the adaptive response in the regenerating limb of the Chinese fire-bellied newt Cynops orientalis immediately after amputation remains unclear. To better understand the adaptive response immediately after limb amputation at the protein level, we used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS methods to analyze changes in the proteome of the regenerating newt limb that occurred 2 h and 8 h after amputation. We identified 152 proteins with more than 1.5-fold change in expression compared to control. GO annotation analysis classified these proteins into several categories such as signaling, Ca 2+ binding and translocation, transcription and translation, immune response, cell death, cytoskeleton, metabolism, etc. Further ingenuity pathway analysis (IPA) showed that several signaling pathways were significantly changed at 2 h and 8 h after amputation, including EIF2 signaling, acute phase response signaling, tight junction signaling and calcium signaling, suggesting these pathways may be closely related to the adaptive response immediately after limb amputation. This work provides novel insights into understanding the molecular processes related to newt limb regeneration immediately after amputation, and a basis for further study of regenerative medicine. KEY WORDS: Cynops orientalis, limb regeneration, proteomic, stress response, cell deathAmong vertebrates, urodele salamanders possess remarkable capability to regenerate appendages from any level of amputation through blastema formation. Subsequently, blastema cells that morphologically resemble mesenchymal stem-like cells self-organize into the amputated limb parts , Nye et al., 2003. Adult newt has already been used as an important model for the limb regeneration studies. The process of limb regeneration can be divided into three major phases: wound healing and dedifferentiation; blastema accumulation and blastema growth; differentiation and morphogenesis (Iten and Bryant, 1973). Following amputation, the wound surface is covered rapidly by epithelial cells, which form the wound epidermis at the end of the stump. More importantly, a specialized epithelium provides signals to the underlying cells of Int. J. Dev. Biol. 59: 487-496 (2015) Abbreviations used in this paper: iTRAQ , isobaric tags for relative and absolute quantitation; IPA, ingenuity pathway analysis.

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Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS

Cited by 224 publications

References 20 publications

Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental Diabetic Neuropathy

Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental Diabetic Neuropathy

Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

iTRAQ-based proteomic analysis of adaptive response in the regenerating limb of the Cynops orientalis newt

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