Blood Peptidome-Degradome Profile of Breast Cancer Yufeng Shen1*, Nikola Toli c´2, Tao Liu1, Rui Zhao 2 , Brianne O. Petritis3, Marina A. Gritsenko 1 , David G. Camp 1 , Ronald J. Moore1, Samuel O. Purvine2, Francisco J. Esteva4, Richard D. Smith1,2* 1 Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 2 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 3 Biological Design Interdisciplinary Graduate Degree Program, Arizona State University, Tempe, Arizona, United States of America, 4 Departments of Breast Medical Oncology and Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America Abstract Background: Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients. Methodology and Principal Findings: The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as .4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin- antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins. Conclusions: Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility. Citation: Shen Y, Tolic´ N, Liu T, Zhao R, Petritis BO, et al. (2010) Blood Peptidome-Degradome Profile of Breast Cancer. PLoS ONE 5(10): e13133. doi:10.1371/ journal.pone.0013133 Editor: Joel M. Schnur, George Mason University, United States of America Received May 14, 2010; Accepted September 6, 2010; Published October 18, 2010 Copyright: 2010 Shen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the National Institutes of Health, National Center for Research Resources (RR18522). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Yufeng.shen@pnl.gov (YS); rds@pnl.gov (RDS) Introduction Breast cancer is the most common malignancy in Western women; in 2009, more than 192,000 women were estimated to be diagnosed and ,40,000 died of this disease in the United States alone [1]. Breast cancer is a heterogeneous disease, and prognosis is determined largely by tumor size, shape, location, and metastasis in addition to molecular characteristics, such as whether the tumor is hormone receptor-positive or -negative, genetic factors, and the rate of cell division [2]. Tumor markers, such as the estrogen receptor, progesterone receptor, and the human epidermal growth factor receptor 2, are routinely employed to assess invasive breast cancers, and in cases of advanced disease, circulating tumor markers CA15-3/BR27-29 or carcinoembryonic antigen may be used to monitor response to therapies [3]. However, there are presently no tumor markers for early detection of the disease in otherwise healthy women [4]. Impeding the development of such markers using proteomics approaches is the expectation that protein targets in blood reflective of cancers, if present, are most likely present at extremely low levels [5]. Cancer progression, invasion, and metastasis require and/or bring about changes to tumor microenvironments that involve protein degradation by cancer degradome proteases [6]. In addition to proteases, the degradome includes protease activators and inhibitors, and degradation substrates [7], [8]. Protease inhibitors account for 5–10% of all cancer-related drugs [9], while protein substrate degradation products afford a potentially rich pool for cancer biomarker discovery [10], [11]. As protein degradation products, intracellular and/or intercellular peptides that constitute the peptidome have been explored for their potential as biomarkers [12]; however, the small pieces of peptide sequences detected and identified are difficult to correlate to available cancer degradomic information, possibly because such small sequences are likely the terminal products of the multi-stage degradation of protein substrates. Herein, we report on degradomic behaviors based upon the analysis of the peptidomes of pooled breast cancer patients and control healthy persons. Our strategy involved comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down, global peptidomic analysis [13]. The results obtained herein demonstrate remarkable variations between the pooled samples that far exceed those evident using conventional bottom- PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13133