Breast Cancer Degradomic View Figure 8. Degradation of complement system MAC key components observed for the tested BCP. (A) For C8, the degradation was solely on the C8a (black color-filled ovals), not on the other fragments (blank-filled ovals). (B) For C9, the degradation was solely on the C9a (black color- filled ovals), not on the other fragments (blank-filled ovals). The red, blue, and black lines along the amino acid sequences have the same significances as for Figure 7. doi:10.1371/journal.pone.0013133.g008 Proteomic analysis Conventional bottom-up approach was used for proteomic analysis of the BCP and HP plasma samples. The samples were depleted using the same procedure as used for preparation of the peptidomic samples. The IgY12 flow-through proteins were denatured and reduced in 50 mM ammonium bicarbonate buffer (pH 8.2), 8 M urea, 10 mM dithiothreitol for 1 h at 37uC, followed by alkylation in 40 mM iodoacetamide for 1 h at room temperature in the dark. The resultant protein mixtures were diluted 10 fold with 50 mM ammonium bicarbonate buffer (pH 8.2), and then sequencing grade modified porcine trypsin (Promega, Madison, WI) was added at a trypsin:protein ratio of 1:50 (w/w). The samples were incubated overnight at 37uC for digestion. The tryptic digest samples were loaded on a 1-mL SPE C18 column (Sigma, St. Louis, MO) and cleaned with 4 mL of 0.1% trifluoroacetic acid/5% acetonitrile. The peptides were eluted from the SPE column with 1 mL of 0.1% trifluoroacetic acid/80% acetonitrile and lyophilized. The peptide samples were stored at 280uC for analysis. The 300 mg of the prepared two tryptic digestion samples were individually reconstituted with 300 mL of 10 mM ammonium formate (pH 3.0)/25% acetonitrile and fractionated by strong cation exchange (SCX) chromatography on a Polysulfoethyl A 200 mm62.1 mm column (PolyLC, Columbia, MD) that was preceded by a 10 mm62.1 mm guard column. The separations were performed on an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA) at a flow rate of 200 mL/min, and with mobile phases that consisted of 10 mM ammonium formate (pH 3.0)/ 25% acetonitrile (A) and 500 mM ammonium formate (pH 6.8)/ 25% acetonitrile (B). After loading 300 mL of each sample separately onto the column, the gradient was maintained at 100% A for 10 min. The peptides were separated using a gradient from 0 to 50% B over 40 min, 50–100% B over 10 min, and then held at 100% B for 10 min. A total of 30 fractions were collected for each sample, and each fraction was dried under vacuum. The fractions for each sample were dissolved in 30 mL of 25 mM NH4HCO3, and 5 mL was used for the capillary LC-MS/MS analyses. Peptides LC-MS/MS analyses were carried out using a custom- built automated capillary LC system coupled online to an LTQ mass spectrometer (Thermo Scientific) via a nanoelectrospray ionization interface manufactured in-house. The LC separations were performed on 650 mm60.075 mm capillary columns containing 3-mm Jupiter C18 bonded particles (Phenomenex, Terrence, CA). The 5 mL of each SCX fraction was loaded onto the column, and the mobile phase was held at 100% A (0.1% formic acid) for 20 min, followed by a gradient from 0 to 70% buffer B (0.1% formic acid in 90% acetonitrile) over 85 min with a flow rate ,500 nL/min. Each full MS scan (m/z 400–2000) was followed by collision-induced MS/MS scans (normalized collision energy setting of 35%) for the 10 most abundant ions. The dynamic exclusion duration was set to 1 min, the heated capillary was maintained at 200uC, and the ESI voltage was held at 2.2 kV. The LC-MS/MS raw data were converted into .dta files using Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo Scientific), and the SEQUEST algorithm (version 27, revision 12) was used to independently search all the MS/MS spectra against the IPI database (version 3.39, released February 2008) with dynamic oxidation on methionine residues and static alkylation on cysteine residues. The false discovery rate (FDR) was estimated based on the reported decoy-database searching methodology [42], and filtering criteria were applied to limit the FDR at the peptide level to ,1% as follows: for charge state +1 peptides, Xcorr .1.5 and DCn.0.05 for fully tryptic peptides and Xcorr .2.5 and DCn .0.16, or Xcorr .3.0 and DCn .0.10, or Xcorr .3.3 and DCn .0.05 for partially tryptic peptides; for charge state +2 peptides, Xcorr .1.5 and DCn .0.16, or Xcorr .1.7 and DCn .0.10, or Xcorr .2.2 and DCn .0.05 for fully tryptic PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13133