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

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.

Comparison of the nuclear proteomes of mammary epithelial cells at different stages of functional differentiation.

The progression of stem cells to proliferating progenitor cells and finally to a quiescent differentiated state is a hallmark of organ development. This process proceeds through distinct steps and is regulated through cell-cell interactions and by systemically and locally acting factors. We have established a cell culture system which recapitulates features of mammary gland development in vitro and allows the comparison of three characteristic differentiation stages. Cell fate decisions relating to proliferation and differentiation are dependent on the function of proteins in the nucleus. Therefore, we have applied proteomic approaches, including 1- and 2-DE coupled with MS and a gel-free system, called protein sequence tag technology (PST), to assess the changes in the nuclear protein composition during differentiation of mammary epithelial cells. We identified about 250 individual proteins which are present in the nucleus of proliferating and functionally differentiated mammary epithelial cells. We functionally categorised the differentially expressed proteins and identified a multitude of proteins that regulate gene expression at the transcriptional or post-transcriptional level. This analysis greatly enriches our global view of the dynamic changes of nuclear proteins during the development of mammary epithelial cells and suggests models for the control of differentiation-specific protein expression.

Protein sequence tags: a novel solution for comparative proteomics.

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.

Characterization of low abundant membrane proteins using the protein sequence tag technology.

About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature. In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions. The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins. The identification of these N-terminal PST peptides is based on LC-MS/MS. The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment. The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins. More interestingly, among these membrane proteins 56% are predicted to be of low abundance. These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.