up proteomics approaches. The present results obtained for the samples carefully selected suggest that the breast cancer patients have increased degradation of functional domains of cancer- relevant proteins, including proteases and inhibitors, extracellular matrix (ECM)-relevant proteins, innate immune system key components, and other protein molecules functioning to suppress cancers. Overall, the present results support the view that the peptidome/degradome is a potentially rich source of makers, support the use of the present top-down proteomic analytical strategies for obtain of the relevant unique information, and highlight the need for more extensive degradomic-peptidomic studies of individual samples, as is enabled by the present work. Results Blood plasma samples for this work were collected under the identical lab protocol from15 breast cancer patients (BCP) (ER positive, Her2 negative; invasive ductal carcinoma; 5 are stage I, 7 are stage II and 3 are stage III) and 15 control healthy persons (HP) of matched age, race, and menopausal status. The blood plasma peptidomes of these samples were isolated without observable discrimination and analyzed in parallel under well- controlled conditions (see Methods below). The BCP and the control HP have very similar sets of plasma proteins (Table S1) identified using a conventional bottom-up proteomics method (i.e., analysis of peptides originating from tryptic digestions of proteome proteins), but show strikingly different degradation patterns (Figure 1) of these proteins that are revealed from analysis of the peptidome peptides. The BCP degradome encompasses 71 protein substrates that generate 839 distinct peptidome peptides, while the HP degradome encompass- es 50 substrates that generate 425 peptides (Table S2), suggesting that the BCP have a more active degradome than the HP. The ratios of the peptidome peptide relative abundances can vary by .4000 fold between the BCP and the HP samples tested (Table S2). The striking degradome differences observed for the BCP also includes selective changes of major high abundance peptidome peptides (Figure 2). Moreover, the degradation observed for the BCP is selective in the degradome substrates. For example, inter-a trypsin inhibitor heavy chain 1 (ITI HC1) produces 22 peptidome peptides in the BCP, but none in the HP; whereas ITI HC4 displays similar selectivity (53 peptides in the BCP, and 48 in the HP). Figure 3 shows that the overall cleavage specificities of degradome proteases active in the BCP and HP are similar, which suggests that the BCP degradome has no proteases that aberrantly cleave protein substrates. However, the cleavage specificity for individual substrates, as illustrated in Figure 4 for ApoA-IV, can vary significantly, which suggests differences in the substrate proteolytic activity between the BCP and the HP examined. Plasmin (Plm) is an extracellular protease that physiologically and pathologically participates in the remolding of tissues and female reproductive organs [14]. The Plm proenzyme plasmino- gen (Plg) is secreted to the extracellular space where it is converted to Plm by removal of the preactivation peptide domain (Glu20- Lys97) through cleavage of Lys97-Val98 (catalyzed by Plm) [15], while the Plm proteolytic activity is controlled by a2-antiplasmin (a2-AP) where Arg403-Met404 and Met404-Ser405 inhibit Plm (and other serine proteases) through formation of stable complexes (e.g., Plm/a2-AP) [16]. The activated Plm has been found to degrade the ECM and its adhesive proteins [17], [18], a required step for cancer invasion and metastasis. Figure 5A shows observations of the differential degradation for Plm-a2-AP system Breast Cancer Degradomic View of the BCP tested. The differential degradation (7:1) for the BCP and control HP occurs for the Plg preactivation peptide (Table S2), which would accelerate conversion of Plg to Plm; while the proteolysis function domain Plm light chain remains unchanged. Degradation of a2-AP is observed at its inhibitory bonds only for the BCP (8:0 for BCP:HP). The implication of Plm in cancer invasion and metastasis has focused on the Plm activation by urokinase-type Plg activator (uPA) through cleaving Lys580- Val581 and releasing the serine protease, i.e., Plm light chain [19]. Our degradomic study demonstrates that the Plm-a2-AP system proteolytic activity for the BCP could also be enhanced by selective degradation of Plg and a2-AP sequence functional domains. The prothrombin (FII)-antithrombin (AT) system is involved in angiogenesis, as well as in cancer invasion and metastasis [20], [21]. This system displays selective and differential degradations (Figure 5B) similar to the Plm-a2-AP system in the BCP (Figure 5A). The increased proteolytic activity of the FII-AT system is in agreement with observations of .100 fold more fibrinopeptide A in the BCP than in the control HP (Table S2). The matrix metalloproteinases (MMPs) [22], a major type of proteases that degrade ECM, are found to have no differences between the BCP and the control HP samples examined from either degradomic or proteomic measurements (Table S3). This is not in contradiction with the hypothesis that the MMPs signature is a poor marker for prognosis of patients with primary breast cancer [23]. Tissue remodeling for cancer invasion and metastasis requires degradation of the ECM [24] that binds surface proximal proteins (Figure S1). It has been found that mediated by TSG-6 [25], the ECM hyaluronan (HA) binds ITI heavy chains (HCs), which make up the complexes IaI [HC1/HC3/LC (LC: ITI light chain)], PaI (HC3/LC), and IaIL (HC2/LC) [26], [27] to inhibit plasmin and other proteases and then protect and stabilize the ECM. Figures 6A–C shows that HC1, HC2, and HC3 are differentially degraded in the BCP tested. The differential degradation occurs to the HC1-3 main chains, preventing them from functioning to form the protein complexes. It is noted that the differential degradation occurs only in HC1-3 that function to protect and stabilize the ECM, but not HC4-5 (Table S2) that have other functions. In addition, only a few peptidome peptides are observed in the BCP for the ECM components, suggesting that these proteins themselves have not undergone broad degradation yet in the ECP examined (Table S2). The complement system is an essential part of the innate immune system [28] and has multiple roles in inhibiting or promoting tumor growth [29]. Figures 7A–C show the differential degradations for complement system key convertases in the BCP. Convertases C3, which occupies a central position in the system, displays differential degradation in the BCP (5-fold more peptides than in the HP, Table S2). The differential degradation occurs to C3b (Figure 7A), but not to the anaphylatoxin C3a released, which could reduce the activation of C5 to C5b that initiates assembly of MAC. The degradation of C3b further occurs to iC3b (C3a, C3b, and C3dg) that contains the TED domain for C3b to attach the target (e.g., tumor cell) surface and enhances antibody-dependent cellular cytotoxicity rather than C3f released during C3b conformation changes [30]. The peptidome peptides observed show that C3dg links to C3a’1, C3c (C3a+C3b) is disrupted, and C3b exists separately during iC3b degradation. These results suggest that iC3b degradation occurs prior to the conformational changes from iC3b to C3dg [30]. Except for MG6a and CUBf, all iC3b domains [30], [31] contribute peptidome peptides, and the peptides generated encompass sequences across C3b multiple ($2) PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13133