Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT)
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...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal Article |
| Published: |
2017
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| Online Access: | http://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5b01154 http://hdl.handle.net/20.500.11937/50901 |
| _version_ | 1848758563581722624 |
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| author | Casey, T. Khan, J. Bringans, S. Koudelka, T. Takle, P. Downs, R. Livk, A. Syme, Robert Tan, Kar-Chun Lipscombe, R. |
| author_facet | Casey, T. Khan, J. Bringans, S. Koudelka, T. Takle, P. Downs, R. Livk, A. Syme, Robert Tan, Kar-Chun Lipscombe, R. |
| author_sort | Casey, T. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T09:45:59Z |
| format | Journal Article |
| id | curtin-20.500.11937-50901 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T09:45:59Z |
| publishDate | 2017 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-509012019-02-19T05:36:19Z Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) Casey, T. Khan, J. Bringans, S. Koudelka, T. Takle, P. Downs, R. Livk, A. Syme, Robert Tan, Kar-Chun Lipscombe, R. 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. 2017 Journal Article http://hdl.handle.net/20.500.11937/50901 10.1021/acs.jproteome.5b01154 http://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5b01154 restricted |
| spellingShingle | Casey, T. Khan, J. Bringans, S. Koudelka, T. Takle, P. Downs, R. Livk, A. Syme, Robert Tan, Kar-Chun Lipscombe, R. Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title | Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title_full | Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title_fullStr | Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title_full_unstemmed | Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title_short | Analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (iTRAQ and TMT) |
| title_sort | analysis of reproducibility for proteome coverage and quantitation using isobaric mass tags (itraq and tmt) |
| url | http://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5b01154 http://hdl.handle.net/20.500.11937/50901 |