Cancer Investigation, iFirst:1–9, 2008 ISSN: 0735-7907 print / 1532-4192 online Copyright c  Informa Healthcare USA, Inc. DOI: 10.1080/07357900701783883 ORIGINAL ARTICLE Breast Cancer Related Proteins Are Present in Saliva and Are Modulated Secondary to Ductal Carcinoma In Situ of the Breast Charles F. Streckfus, 1 Otilia Mayorga-Wark, 2 Daniel Arreola, 1 Cynthia Edwards, 1 Lenora Bigler, 1 William P. Dubinsky, 2 University of Texas Health Science Center—Dental Branch, Houston, Texas, USA, 1 University of Texas Health Science Center—Medical School, Houston, Texas, USA 2 ABSTRACT Objective: The objective of this study was to determine if protein-by-products secondary to cancer related oncogenes appear in the saliva of breast cancer patients. Methods: Three pooled (n = 10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women di- agnosed with a benign breast tumor and the other one pooled specimen was from women diagnosed with ductal carcinoma in situ (DCIS). Differential expression of proteins was mea- sured by isotopically tagging proteins in the tumor groups and comparing them to the healthy control group. Experimentally, saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. Results: The results of the salivary analyses in this population of patients yielded approximately 130 proteins in the saliva specimens. Forty-nine proteins were differentially expressed between the healthy control pool and the benign and cancer patient groups. Conclusions: The study sug- gests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to DCIS. Additionally, there may be salivary protein profiles that are unique to both DCIS and fibroadenoma tumors. INTRODUCTION Proteomics was originally defined to represent the analysis of the entire protein component of a cell or tissue (1), but now it encompasses the study of expressed proteins, including iden- tification and elucidation of the structure-function relationship under healthy conditions and disease conditions. In combination with genomics, proteomics can provide a holistic understanding of the biology underlying disease processes. Information at the Keywords: Saliva, LC-MS/MS, isotope labeling, breast cancer Correspondence to: Charles F. Streckfus, DDS, MA, FAAOM, FAGD UTHSC - Dental Branch 6516 M.D. Anderson Blvd., Rm 4.133 Houston, Texas 77030 tel: 713-500-4531; fax: 713-500-4372. e-mail: charles.streckfus@uth.tmc.edu. level of the proteome is critical for understanding the function of specific cell types and their role in health and disease (1, 2). Protein expression and function are subject to modulation through transcription as well as through posttranscriptional and translational events. Multiple RNA species can result from one gene through a process of differential splicing. Additionally, there are more than 200 post-translation modifications that pro- teins could undergo that affect function, protein-protein and nuclide-protein interaction, stability, targeting half-life, and so on (3–5), all contributing to a potentially large number of pro- tein products from one gene. Identifying and understanding these changes are the underlying themes in proteomics (6–8). Technological advancements have benefited proteomic re- search to the point where saliva is now being assayed for protein content using the latest available proteomic technology (9). The investigators opted to explore saliva as a diagnostic fluid for two reasons:collection of saliva is a non-invasive procedure that can be conducted in any environment requiring no special skills of equipment and the physiology of the oral cavity is such that the 1