Figure 5. Selective degradation of function domains of protease systems observed for the tested BCP. (A) For Plm-a2- AP system, Plg degrades on its preactivation peptide; while a2-AP on the inhibitory function bonds. (B) For FII-AT system, FII degrades on its heavy chain, not on its proteolytic function domain; while AT degrades on its inhibitory function bonds to prevent formation of thrombin- antithrombin (TAT) complex. The red lines along the sequences represent the peptidome peptides solely observed from the BCP; the differential degradation is represented by the numbers (red for the BCP and blue for the HP) of different peptides observed. doi:10.1371/journal.pone.0013133.g005 Caucasians, 3 Hispanics and 1 Asian/Pacific Islander; 8 premenopausal and 7 postmenopausal; aged from 30–78 years old (median age is 48 years old). The 15 control healthy subjects were carefully selected to have matched age, race, and menopausal status as the BCP. Blood samples collected in EDTA tubes were first centrifuged at 1,500–1,600 g for 15 min at 4uC, and plasma (upper phase) was transferred to another EDTA tube and centrifuged at 14,000 g for 15 min at 4uC to remove any remaining cellular material. The top 90% of plasma was then Breast Cancer Degradomic View transferred into cryovials with approximately 0.5 ml plasma in each and stored frozen at 280uC until further use. Degradomic-peptidomic analysis The method reported [13] was used for peptidomic analysis. Briefly, prior to analysis, 10 mL of Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Inc., Rockford, IL) was added into every 1 mL of the plasma samples. The two pooled blood plasma samples of the BCP and the control HP from the matched BCP and HP were depleted of its 12 high-abundance proteins using a 12.7679.0 mm IgY12 LC10 affinity chromatography (AC) column (Beckman Coulter, Fullerton, CA). The AC-depleted plasma samples were separated using a size exclusion chromatog- raphy (SEC) column (Superdex 200 10/300 GL, GE Healthcare, Piscataway, NJ), and species ,20 kDa were collected (based on the calibration with various sizes of standard proteins) and denatured (1 h at 37uC with 8 M urea). The resulting solution was buffer exchanged to 50 mM NH4HCO3 (pH 8.0) in Amicon Ultra-15 filter (3 kDa nominal MW limit, Millipore, Billerica, MA). The plasma peptidomes of these samples were isolated without observable discrimination (Figure S3). The AC/SEC- isolated peptidome components (50 mg of each sample) were separated using the high-resolution liquid chromatography (HRLC). The HRLC was performed on a 1000 mm60.100 mm i.d. fused silica capillary column containing 3-mm porous (300 A˚ size) C4-bonded silica particles (Sepax Technologies, Inc. Newark, DE) with the mobile phase gradient from A (acetonitrile/H2O/ acetic acid, 10:90:0.2, v/v/v) to B (acetonitrile/isopropyl alcohol/ H2O/acetic acid/trifluoroacetic acid, 60:30:10:0.2:0.1, v/v/v/v/ v). Two HRLC separation runs were for each sample with gradients of 14,000 and 16000 min, respectively. The HRLC- separated components were detected using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) under the conditions as follows: AGC targets of 16106 and 36105, respectively, for the Fourier transform mass spectrometry (FT MS) and FT tandem MS (MS/MS), 60K resolution for acquirement of the spectra, 400#m/z#2000 for a survey scan followed by FT MS/MS of the 5 most intense ions from the survey scan, 35% normalized collision energy employed for CID-FT MS/MS, a duration cycle of 30 s for the dynamic exclusion, 3 micro scans selected for FT MS and MS/MS measurements of the two HRLC-FT MS/MS analyses of each sample. The unique sequence tags (UStags) method [13] was used to process the FT MS/MS data using the ICR2LS developed in-house (http://ncrr. pnl.gov/software/), and only unique sequences from the IPI human sequence database (ipi.HUMAN.v3.39 downloaded from ftp://ftp.ebi.ac.uk/pub/databases/IPI/) were used for identifica- tion of the peptidome peptides. The identification false rate was estimated using a decoy dataset that was constructed by reversing each protein sequence in the human sequence database; no random hits were obtained using the UStags as the identification criteria. The identification of disulfide bond-containing peptides was completed with the method reported elsewhere [40]. The protein annotations and information from EBI (http://www.ebi. ac.uk/) and Swiss-Prot (http://ca.expasy.org) were used to describe the proteins (substrates) identified in this work. Quantitation of peptidome peptides was achieved through extraction of the FT- measured precursors of the FT-MS/MS Spectra. ICR2LS was used to deisotope high-resolution spectra and produce lists of neutral masses for both precursor MS and MS/MS spectra. Functions were built specifically for this study to isolate MS spectra only and merge re-enumerated spectra to form a single continuous dataset. ICR2LS was also used to assign absolute intensity (abundance) values for the deisotoped masses PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13133