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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 37C 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.