Protein Expression Profiling of Breast Cancer Cells by Dissociable Antibody Microarray (DAMA) Staining

Abstract

Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer. Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer. Protein microarrays have recently been attracting great attention for their potential use in high throughput studies of protein function (1Emili A.Q. Cagney G. Large scale functional analysis using peptide and protein arrays.Nat. Biotechnol. 2000; 18: 393-397Crossref PubMed Scopus (231) Google Scholar, 2de Wildt R.M. Mundy C.R. Gorick B.D. Tomlinson I.M. Antibody arrays for high-throughput screening of antibody-antigen interactions.Nat. Biotechnol. 2000; 18: 989-994Crossref PubMed Scopus (579) Google Scholar, 3Kadadek T. Protein microarrays: prospects and problems.Chem. Biol. 2001; 8: 105-115Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 4Macbeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (748) Google Scholar, 5Bertone P. Snyder M. Advances in functional protein microarray technology.FEBS J. 2005; 272: 5400-5411Crossref PubMed Scopus (142) Google Scholar, 6Hall D.A. Ptacek J. Snyder M. Protein microarray technology.Mech. Aging Dev. 2007; 128: 161-167Crossref PubMed Scopus (219) Google Scholar). The ultimate goal of developing this technology is to construct ordered arrays of individual proteins for biochemical study at the molecular level. A number of different sources of peptides and proteins have been used for protein microarray manufacture, including synthetic peptides (7Roep B.O. Hiemstra H.S. Schloot N.C. De Vries R.R. Chaudhuri A. Behan P.O. Drijfhout J.W. Molecular mimicry in type 1 diabetes: immune cross-reactivity between islet autoantigen and human cytomegalovirus but not Coxsackie virus.Ann. N. Y. Acad. Sci. 2002; 958: 163-165Crossref PubMed Scopus (33) Google Scholar, 8Collett J.R. Cho E.J. Ellington A.D. Production and processing of aptamer microarrays.Methods. 2005; 37: 4-15Crossref PubMed Scopus (121) Google Scholar), recombinant proteins (9MacBeath G. Schreiber S.L. Printing proteins as microarrays for high-throughput function determination.Science. 2000; 289: 1760-1763Crossref PubMed Google Scholar, 10Ge H. UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protei