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Supplemental Methods Peptide selection, synthesis, quantitation and handling Synthetic tryptic proteotypic peptides from human protein C inhibitor (PCI) and soluble transferrin receptor (sTfR) were used. Peptides were selected according to criteria previously described (7). Both unlabeled and stable isotope labeled versions of peptides were synthesized by JPT Peptide Technologies GmbH (Berlin, Germany). Resin-bound custom peptide synthesis was performed using Fmoc-protection strategy and HPLC-MS analytical methods were used for quality control by the vendor. With the stable isotope versions of the peptides a mass increment was added in each case through the use of labeled C-terminal arginine (+ 10 amu) or lysine (+ 8 amu) providing mass shifts of m/z = + 5 or + 3 for typical doubly-charged peptide ions. The tryptic PCI peptide (positions 239-248, approximately in the middle of the primary sequence of the protein) was synthesized and its corresponding stable isotope standard (SIS) peptide was produced with a labeled arginine residue: EDQYHYLLDR* (+10 amu). In addition, a stable isotope labeled peptide was designed as a control for tryptic digestion. This “winged” peptide was constructed with 4 extra amino acids at each end (underlined portions of the sequence below) and contained two tryptic cleavage sites. Tryptic digestion of this peptide releases a smaller peptide that is identical in sequence to the endogenous peptide but with different isotopic configuration: MMSREDQYHYL*L*DR*NLSC EDQYHYL*L*DR*. The released peptide is +24 amu with respect to the endogenous peptide and +14 amu with respect to the SIS peptide. As a control peptide in a 2-plex assay, we used a tryptic surrogate peptide from soluble transferrin receptor (sTfR): GFVEPDHYVVVGAQR. The corresponding SIS peptide contained a labeled arginine at the C-terminus: GFVEPDHYVVVGAQR* (+10 amu). All peptides were of greater than 90 % purity as determined by HPLC. Upon receipt from the vendor, peptide stocks were adjusted to approximately 10 nmol/L (based on dry weight) in 30 % acetonitrile/0.1 % formic acid. However, stock concentrations determined by peptide weight are inaccurate due to differing hygroscopic properties, hydrophobicity and solubilities of peptides, thus aliquots of these initial stocks were sent for quantitation by amino acid analysis (AAA; Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, Ontario). Using AAA data, the peptide stocks were then readjusted to 10 nmol/L. Peptides were diluted in 30 % acetonitrile/0.1 % formic acid to 10 pmol/L immediately prior to use and were stored for short periods (2 weeks or less) at 4 °C in solution phase. After thawing and/or just before use, all peptides were analyzed by MALDI-TOF-MS to determine their integrity and to assess the presence of altered forms. Derivation and selection of high affinity anti-peptide monoclonal antibodies Rabbit anti-peptide monoclonal antibodies (RabMAbs) were derived against the PCI and sTfR peptides (SISCAPA Assay Technologies Inc., Washington DC, in collaboration with Epitomics Inc., Burlingame, CA). To select high affinity anti-peptide RabMAbs, screening of ELISA positive hybridoma supernatants was performed at the University of Victoria using a combination of surface plasmon resonance and MALDI immunoscreening (13). This process allowed selection of anti-peptide antibodies with nanomolar or better affinity, capable of binding low abundance peptides from solution and retaining them through extensive washing steps that minimize non-specific background binding of peptides. Specifically, the mAb specific for the PCI peptide had an affinity of 0.09 nM (half off-time of 68 min) and the mAb specific for the sTfR peptide had an affinity of 1.8 nM (half off-time of 54 min). Digestion Protocol An “addition-only” digestion protocol (8) was used to digest 100-1000 L of the serum samples. Briefly, aliquots of a denaturation mix of urea, tris(2- carboxyethyl)phosphine (TCEP) and Tris buffer (pH 8.1) were lyophilized, such that upon reconstitution with a known volume of serum, yielded final concentrations of 9 M, 0.05 M, and 0.2 M respectively. Neat plasma or sera were spiked with the digestion control peptide at 50 fmol/L and then added to the lyophilized denaturation mix, followed by 30 min incubation at room temperature. To prevent reformation of disulfide bonds, iodoacetamide was added at a 1.5 molar excess over cysteine residues (26 mM in serum proteins) followed by incubation of the sample for 30 min in the dark. The reaction mixture was diluted to a final urea concentration of 1 M before adding trypsin (Worthington Cat. No. LS003740) at a 1:20 ratio of enzyme to substrate. Samples were incubated 16 h at 37C before adding a 2-fold excess of tosyl-L-lysine chloromethyl ketone (TLCK) to eliminate tryptic activity. The digested samples were purified using solid phase extraction (SPE) columns (Oasis HLB 6 cc cartridges; Waters, Milford, MA) according to the manufacturer’s instructions. SISCAPA-MALDI Assay Development All SISCAPA experiments requiring peptide enrichment were performed using a magnetic bead-handling robot (KingFisher 96; Thermo Electron Corporation, Vantaa, Finland). In all experiments, each well of the reaction plate contained the digest resulting from 10 L of specimen spiked with 500 fmol/uL of corresponding heavy (SIS) peptides and diluted 1/10 (v/v) in PBS/0.03% CHAPS. Custom made, prototype magnetic protein G coated beads (1.0 micron diameter; Invitrogen, Oslo, Norway) were used to capture 1 g/well of specific monoclonal antibodies. Standard 2.4 micron magnetic DynabeadsTM Protein G (Cat No. 10004D; Novex-Life Technologies) can also be used. In this standard procedure the robot transferred the antibody-coated beads to the reaction plate where antibodies captured the peptide analyte of interest out of the digest during the 1-h incubation. The bead-antibody complex was then transferred through three consecutive wash steps to reduce the non-specific background. The first two wash plates contained 250 L of PBS/0.03% CHAPS per well and the third wash plate contained 250 L of 75% ACN in PBS/CHAPS per well. The total elapsed time during the wash steps was approximately 10 min (i.e., shorter than the half off-time of the anti-peptide antibodies). Finally, the bead-antibody complex reached the elution plate where the target peptides were eluted in 13 L of 0.1% formic acid. In the experiments reported here, half of the eluted sample was spotted onto MALDI targets for analysis as described below. Negative controls and analyte specificity For each run we included two negative controls to ensure the identity of the analytes being measured. A “no-antibody control” was used, which controlled for interferences in both the light and heavy channels since these peptides should only be present if they had been enriched by the specific antibodies. A “PCI-deficient plasma control” was used, which controlled for interferences in the light channel when the antibody was present but the analyte had been depleted. MALDI-TOF analysis of eluted peptides Peptides were analyzed using two MALDI-TOF mass spectrometers: a 4800 MALDI-TOF/TOFTM Analyzer with 4000 series Explorer v3.5 software (AB Sciex, Framingham, MA) and a linear-mode only instrument called the microflexTM LT (Bruker Daltonics, Billerica, MA). In both cases, we first dried 6 L of the SISCAPA enriched peptide eluate on the MALDI targets and then spotted 1 L of the α-Cyano-4hydroxycinnamic acid (CHCA) matrix (5 mg/mL CHCA and 1 mg/mL ammonium citrate dibasic in 70% ACN/0.1% FA) onto each sample. On the AB 4800 instrument, acquisition of data was automated in the 800-4000 Da mass range and in the positive-ion reflector mode with a laser intensity of 3400 at 1000 shots/spectrum. The ratio of the intensity of the most abundant monoisotopic peak from the endogenous analyte (m/z 1351) to the corresponding peak for the stable isotope peptide (m/z 1361) was used for quantitation purposes. Data Explorer v4.2 (AB Sciex, Framingham, MA) was used for data analysis. For MS/MS analysis of the specific peptide precursors, the collision energy was set to 2 kV and the relative precursor mass window was set at 300 (FWHM). MS/MS spectra were collected with collision-induced dissociation (CID) turned on and 1250 shots/spectrum. To verify the sequence of the peptide, MS/MS spectra were manually examined using the MS-Product tool from the ProteinProspector software v5.5.0 (University of California, San Francisco, CA). On the microflex LT instrument, the acquisition was performed in the linear mode only since the microflex LT only offered this option. Automated acquisition was performed with parameters optimized to give at least partially resolved isotopic envelopes up to m/z 1600. For each sample, 500 to 800 laser shots were performed and counts averaged. The SNAP algorithm was used to determine the ratio of the whole isotopic envelope for the endogenous analyte to that of the stable isotope peptide for quantification purposes. FlexAnalysis v3.4 (Bruker Daltonics, Billerica, MA) was used for data analysis. Subjects All study subjects had histologically confirmed adenocarcinoma of the prostate with no restrictions on Gleason scores or PSA concentrations. Treatment regimens were at the discretion of the treating physician. All blood samples were collected with informed consent and approval from the Research Ethics Board of the BC Cancer Agency. Fifty-one patients with non-metastatic prostate cancer were recruited at the BC Cancer Agency in Victoria, BC, Canada. Pretreatment serum was obtained at the first patient visit and subsequent samples were collected every 3 months during treatment and every 6 months after the completion of treatment. Study blood draws were organized to coincide with regularly scheduled PSA tests. Medical records were retrospectively reviewed and the Phoenix definition of biochemical recurrence was used. Linearity study by mixing different samples Linearity of response and recovery was also tested by mixing equal volumes of the PCI-deficient plasma and a pooled plasma sample that was known to have endogenous analyte. The three samples (PCI-deficient plasma, pooled plasma and the 1:1 mixture) were each digested three times and the PCI peptide antigen was measured in all samples. Bilirubin and hemoglobin interferences To test for possible interferences from bilirubin and hemoglobin, pooled human plasma was spiked with intact bilirubin and hemoglobin at different levels (10-40 μg/mL). Each sample was digested three times and the PCI analyte was measured.