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    Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT)

    Access Status
    Fulltext not available
    Authors
    Casey, T.
    Khan, J.
    Bringans, S.
    Koudelka, T.
    Takle, P.
    Downs, R.
    Livk, A.
    Syme, Robert
    Tan, Kar-Chun
    Lipscombe, R.
    Date
    2017
    Type
    Journal Article
    
    Metadata
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    Citation
    Casey, T. and Khan, J. and Bringans, S. and Koudelka, T. and Takle, P. and Downs, R. and Livk, A. et al. 2017. Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT). Journal of Proteome Research. 16: pp. 384-392.
    Source Title
    Journal of Proteome Research
    DOI
    10.1021/acs.jproteome.5b01154
    Additional URLs
    http://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5b01154
    ISSN
    1535-3893
    School
    Centre for Crop Disease Management
    URI
    http://hdl.handle.net/20.500.11937/50901
    Collection
    • Curtin Research Publications
    Abstract

    This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be =69% for technical duplicates and =57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools?ProteinPilot, Mascot, and Proteome Discoverer?the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.

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