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Differential Proteomic Analysis of Acute Contusive Spinal Cord Injury in Rats Using iTRAQ Reagent Labeling and LC–MS/MS

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Abstract

In this experimental study, differential labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) proteomic approach was used to investigate differences in the proteome of rat spinal cord at 24 h following a moderate contusion injury. Spinal cord protein samples from the injury epicenter (or from sham controls) were trypsinized and differentially labeled with iTRAQ isotopic reagents. The differentially labeled samples were then combined into one sample mixture, separated by LC, and analyzed using MS/MS. Proteins were quantified by comparing the peak areas of iTRAQ reporter fragment ions in MS/MS spectra. The outcome of this analysis revealed that proteins involved in ubiquitination, endocytosis and exocytosis, energy metabolism, inflammatory response, oxidative stress, cytoskeletal disruption, and vascular damage were significantly altered at 24 h following spinal cord injury (SCI). This study demonstrates the utility of the iTRAQ method in proteomic studies and provides further insights into secondary events that occur during acute times following SCI.

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Abbreviations

LC:

Liquid chromatography

MS:

Mass spectrometry

iTRAQ:

Isobaric tag for relative and absolute quantitation

CI %:

Confidence interval %

MW:

Molecular weight

SCI:

Spinal cord injury

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Acknowledgments

The authors gratefully acknowledge Dr. Melanie L. McEwen for technical support. This work was supported by the Craig H. Neilsen Foundation, the Morton Cure Paralysis Foundation, and an endowment from Cardinal Hill Rehabilitation Hospital.

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Authors and Affiliations

  1. Departments of Physical Medicine and Rehabilitation, Anatomy and Neurobiology, and the Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY, 40536, USA

    Anshu Chen, RangaswamyRao Ravikumar, Nishant P. Visavadiya & Joe E. Springer

  2. Laboratory of Molecular Psychiatry, Abraham Ribicoff Research Facilities, Yale University, New Haven, CT, 06508, USA

    Anshu Chen

  3. Applied Biosystems, Framingham, MA, 01701, USA

    Shixin Sun

  4. Cardinal Hill Rehabilitation Hospital, Lexington, KY, 40504, USA

    Joe E. Springer

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  1. Anshu Chen
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  2. Shixin Sun
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  3. RangaswamyRao Ravikumar
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  4. Nishant P. Visavadiya
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  5. Joe E. Springer
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Correspondence to Joe E. Springer.

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Chen, A., Sun, S., Ravikumar, R. et al. Differential Proteomic Analysis of Acute Contusive Spinal Cord Injury in Rats Using iTRAQ Reagent Labeling and LC–MS/MS. Neurochem Res 38, 2247–2255 (2013). https://doi.org/10.1007/s11064-013-1132-y

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  • DOI: https://doi.org/10.1007/s11064-013-1132-y

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