Liquid chromatography tandem-mass spectrometry (LC-MS/MS)- based methods such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT) have been shown to provide overall better quantification accuracy and reproducibility over other LC-MS/MS techniques. However, large scale projects like the Clinical Proteomic Tumor Analysis Consortium (CPTAC) require comparisons across many genomically characterized clinical specimens in a single study and often exceed the capability of traditional iTRAQ-based quantification. Additionally, efforts have been made to achieve reliable iTRAQ quantification results by devising a strategy to minimize the impact of experimental factors on the selection of protein candidates.
CPTAC investigators evaluated the long-term reproducibility of an iTRAQ-based quantitative proteomics strategy using a basal and luminal subtype of patient-derived xenograft breast cancer as well as one channel as a reference sample to normalize across all datasets. As reported in the Journal of Proteome Research, investigators analyzed the xenograft samples by 2D-LC-MS/MS. After LC-MS/MS data collection spanning seven months, all datasets were used to evaluate a standard data analysis pipeline to determine the long-term reproducibility of the experimental procedure.
After confidence score filtering and statistical analysis, the CPTAC investigators observed consistent quantification results from the 2D LC-MS/MS datasets generated over the seven-month time frame. The strategy implemented in the study, in combination with quality control methods, standard operating procedures, and identification-independent quality metrics, could improve the quality of proteomic data, especially for large scale clinical studies.
The study is published in the Journal of Proteome Research.
To learn more about CPTAC – click here.