Breast Cancer Proteomics – Differences in Protein Expression between Estrogen Receptor-Positive and -Negative Tumors Identified by Tandem Mass Tag Technology

Abstract

Background: Proteomic analysis has become an effective tool in breast cancer research. In this study, we applied the new gel-free tandem mass tag (TMT) reference method for the first time in breast cancer. Materials and Methods: Proteomic analysis was used to compare 10 estrogen receptor (ER)-positive and 10 ER-negative samples. The results of the proteomic approach were validated by Western blot, immunohistochemistry and gene expression analysis. Results: 17 proteins with significant differences in expression were identified. 13 proteins were overexpressed in ER-negative tumors and 4 were overexpressed in ER-positive samples. All these proteins were characterized by relatively high cellular abundance. Conclusions: Our results demonstrate that the gel-free TMT approach allows the quantification of differences in protein expression levels. Further improvement of the sensitivity by subfractionation of the tissue should allow also the identification of low-abundance proteins and might lead to the use of this method in breast cancer research.

Early second-trimester plasma protein profiling using multiplexed isobaric tandem mass tag (TMT) labeling predicts gestational diabetes mellitus

“…This method is successfully used to screen for novel biomarkers in periodontal disease, colorectal cancer tissue and so on [18,19]. Previous study found that TMT labeling was a powerful tool for breast cancer proteomics to find differences in protein amount between estrogen receptor-positive and estrogen receptor-negative tumors [20]. These indicating that this approach is applicable to quantitative proteomic studies.…”

TMTpro: Design, Synthesis, and Initial Evaluation of a Proline-Based Isobaric 16-Plex Tandem Mass Tag Reagent Set

“…Since we first introduced the concept in 2003, isobaric peptide tagging for shotgun proteomics has become an important technique in quantitative mass spectrometry with a growing number of powerful and far-reaching applications in areas such as biomarker discovery, , temporal regulation of proteome dynamics, drug target identification/validation, spatial proteomics, protein turnover, proteogenomics, − and drug/protein interaction studies . While isobaric tagging has established itself as an accurate, reliable, and sensitive quantification and multiplexing technique, there is still demand for increased isobaric multiplexing capacity.…”

Reagents for Isobaric Labeling Peptides in Quantitative Proteomics

“…Commercially available labeling reagents such as iTRAQ and TMT are activated NHS esters that are easily hydrolyzed once dissolved in solution, and are completely lost labeling activity within a short period. − Once vials of these reagents are opened, all of them have to be used in one experiment. Thus, the hydrolysis properties would restrict an experimental design and cause the increased cost in experiments.…”

“…There are several approaches based upon mass spectrometry for quantitative proteomics, including label-free, MS1- and MS2-based labeling methods . Isobaric tag labeling methods that belong to MS2-based approaches are widely used. − Compared with label-free data dependent analysis (DDA) methods, the analysis time and run-to-run variations in the isobaric labeling are greatly reduced because the labeled peptides from multiple samples are mixed together before the LC–MS/MS assay. The sensitivity of peptide detection is greatly enhanced because the MS/MS signal(s) for a peptide is contributed by multiple samples so that its corresponding signal intensity is strengthened.…”