Creative Proteomics Blog

In the past decade, genomics research has systematically mapped genetic changes in human cancer, however, we still know little about the direct effects of these changes on the proteome. Proteomics studies of the NIH Clinical Proteomic Tumor Analysis Consortium (CPTAC) have shown that the integration of proteomic and phosphorylated proteome data with genomic data can improve the identification of tumor pathways.

However, solid tumor tissue is much more complex in composition than cell lines, containing at least epithelial, stroma, and blood components. Researchers will need more complex and time-consuming protocols to deal with solid tumor tissue in order to detect low-abundance proteins such as oncogenes and tumor suppressors. Therefore, it is necessary to reduce the complexity of the sample before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Based on this purpose, researchers at the proteomics platform of Broad Institute recently released an optimized new process which can enable rapid and repetitive analysis and quantification of tumor proteins, related result is published on Nature Protocols.

'We introduced an optimized process that utilizes a 10-label tandem mass tag (TMT) for multiplex analysis and relative quantification so as to enable an overall proteome and phosphorylated proteome analysis of the tissue or cell line,' the authors write in the article.

Compared to the 4-label iTRAQ method, this method has a three-fold increase in throughput and improves reproducibility between the laboratories. This process was tested in three independent laboratories, two of which were derived from a xenograft model. Proteomic analysis shows excellent depth and is capable of distinguishing proteins derived from tumor cells.

The researchers also claimed that, 'In the 10,000 proteins quantified per sample, we can distinguish 7,700 human proteins derived from tumor cells, and 3,100 mouse proteins derived from the surrounding matrix and blood.'

The entire process includes collection of tissue, peptide processing, LC-MS/MS, and data analysis. And the complete analysis of 10 samples includes sample processing and data output which can be achieved in 10 days with the application of a single LC-MS/MS instrument. The whole process for the analysis of 100 samples can be completed in about 4 months.

The researchers point out that this improved approach has significantly accelerated research compared to past research which took 9 months to complete the quantification and analysis of the proteome and phosphorylated proteome of 100 samples. They also concluded that, 'At present, high quality, deep and repeatable data from within and between laboratories will help integrate mass spectrometry-based proteomics analysis with proteomic-genomics data, thereby offering new biological insights.'

Reference:
1.Mertins P, Tang L C, Krug K, et al. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry. Nature protocols, 2018: 1.