title: 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.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Paweena
authors.initials: P
authors.lastname: Kreunin
authors.forename: Virginia
authors.initials: V
authors.lastname: Urquidi
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
authors.forename: Steve
authors.initials: S
authors.lastname: Goodison
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology; D002277:Carcinoma / Q000737:chemistry* / Q000473:pathology; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D010641:Phenotype; D020543:Proteome / Q000032:analysis; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D020543:Proteome