Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

Abstract

For most cancer cell types, the acquisition of metastatic ability leads to clinically incurable disease. The identification of molecules whose expression is specifically correlated with the metastatic spread of cancer would facilitate the design of therapeutic interventions to inhibit this lethal process. In order to facilitate metastasis gene discovery we have previously characterized a pair of monoclonal cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immune-compromised mice. In this study, serum-free conditioned media was collected from the cultured monoclonal cell lines and a mass mapping technique was applied in order to profile a component of each cell line proteome. We utilized chromatofocusing in the first dimension to obtain a high resolution separation based on protein pI, and nonporous silica reverse-phase high performance liquid chromatography was used for the second dimension. Selected proteins were identified on the basis of electrospray ionization time of flight mass spectrometry (ESI-TOF MS) intact protein mapping and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting. Using this approach we were able to map over 400 proteins and plot them as a 2-D map of pI versus accurate M r . This was performed over a pI range of 4.0-6.2, and a mass range of 6-80 kDa. ESI-TOF MS data and further analysis using MALDI-TOF MS confirmed and identified 27 differentially expressed proteins. Proteins associated with the metastatic phenotype included osteopontin and extracellular matrix protein 1, whereas the matrix metalloproteinase-1 and annexin 1 proteins were associated with the non-metastatic phenotype. These findings demonstrate that the mass mapping technique is a powerful tool for the detection and identification of proteins in complex biological samples and which are specifically associated with a cellular phenotype.

Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients

“…14 Increased expression of cSHMT has been found in metastatic human breast carcinoma cells MDA-MB-435 compared to nonmetastatic cells. 17 Whether cSHMT function is affected in breast and ovarian cancers remains to be investigated. Tbx3, by contrast, is involved in development of mammary gland; mutations or lack of Tbx3 result in mammary hypoplasia and even in lack of mammary glands.…”

Breast tumor metastasis: analysis via proteomic profiling

“…We have applied these methods to the profiling of first the secreted proteome, and second, the total cellular protein component of the MDA-MB-435 metastasis model [60][61][62]. Within the serum-free conditioned media obtained from the cell lines, we were able to detect over 400 unique, intact proteins and plot them as a 2D map of pI versus accurate molecular mass (Figure 2).…”

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

“…The quantitative proteomic profiling of proteins using multidimensional chromatography has also been performed (reviewed in [20]). A number of proteomic studies have been performed utilising such techniques [21][22][23]. More recently, multidimensional protein separations have been performed integrating both 'top down' and 'bottom up' approaches for protein identifications [24].…”