Addressing accuracy and precision issues in iTRAQ quantitation

Karp, Natasha A., Huber, Wolfgang, Sadowski, Pawel G., Charles, Philip D., Hester, Svenja V., & Lilley, Kathryn S. (2010) Addressing accuracy and precision issues in iTRAQ quantitation. Molecular and Cellular Proteomics, 9(9), pp. 1885-1897.

View at publisher (open access)


iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Impact and interest:

239 citations in Scopus
223 citations in Web of Science®
Search Google Scholar™

Citation counts are sourced monthly from Scopus and Web of Science® citation databases.

These databases contain citations from different subsets of available publications and different time periods and thus the citation count from each is usually different. Some works are not in either database and no count is displayed. Scopus includes citations from articles published in 1996 onwards, and Web of Science® generally from 1980 onwards.

Citations counts from the Google Scholar™ indexing service can be viewed at the linked Google Scholar™ search.

Full-text downloads:

4 since deposited on 02 Jan 2014
4 in the past twelve months

Full-text downloads displays the total number of times this work’s files (e.g., a PDF) have been downloaded from QUT ePrints as well as the number of downloads in the previous 365 days. The count includes downloads for all files if a work has more than one.

ID Code: 65593
Item Type: Journal Article
Refereed: Yes
Additional Information: This research was originally published in Molecular and Cellular Proteomics. Natasha A. Karp, Wolfgang Huber, Pawel G. Sadowski, Philip D. Charles, Svenja V. Hester and Kathryn S. Lilley. Addressing Accuracy and Precision Issues in iTRAQ Quantitation. Molecular & Cellular Proteomics. 2010; 91885-1897. © the American Society for Biochemistry and Molecular Biology.
DOI: 10.1074/mcp.M900628-MCP200
ISSN: 1535-9484
Divisions: Current > Institutes > Institute for Future Environments
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Deposited On: 02 Jan 2014 02:18
Last Modified: 28 Jun 2017 14:45

Export: EndNote | Dublin Core | BibTeX

Repository Staff Only: item control page