Plasma samples
Plasma samples were obtained from a cohort of 64 subjects from the outpatient PRA-screening program of St. Paul’s Hospital (SPH) Laboratory, selected to span the analytical range of the RIA assay (0.05-2.00 ng/L/s using pH = 7.4 incubation) [6]. Samples were collected into pre-chilled EDTA tubes, centrifuged at 4°C, and frozen at −20°C for at most 10 days. Specimens were thawed for 5 min in a room temperature (RT) water bath and immersed in ice-water prior to RIA [6] and LC/ESI-MS/MS analysis, using a variation of our previous method [31]. Samples were then refrozen and maintained at −80°C until iMALDI analysis at the University of Victoria (UVic) - Genome BC Proteomics Centre. Because freeze-thaw cycles can cause elevation of PRA due to prorenin cryoactivation, samples spent minimal times at refrigeration temperatures where cryoactivation is greatest [3].
This study was approved by the research ethics board of the University of British Columbia and SPH.
Chemicals and reagents
RIA and LC/ESI-MS/MS
RIA analysis used a rabbit polyclonal anti-angiotensin antiserum prepared according to a published protocol [39]. Purified Ang-I was purchased from Proteochem. BSA and glacial acetic acid were purchased from EMD Millipore. Radiolabeled Ang-I (Human, [125I]Tyr4-, 10 μCi(370 kBq), 125I-Ang-I) tracer was purchased from Perkin Elmer. Tris, EDTA, formic acid (FA) and PMSF were purchased from Sigma Aldrich. Dextran was purchased from MP Biomedical, and activated charcoal and methanol were purchased from Fisher Scientific. Stable-isotope labelled internal standard (SIS) Ang-I (DR*VYIHPFHL, +10 Da) was prepared by the UVic - Genome BC Proteomics Centre. Briefly, C-terminal [13C]/[15N] labeled tryptic peptides were synthesized using an N-(9-fluorenyl)methoxycarbonyl) procedure on a Prelude peptide synthesizer (Protein Technologies). Peptides were purified by HPLC, purity was confirmed by MALDI-TOF-MS, characterization was done by capillary zone electrophoresis at the University of British Columbia (Vancouver, Canada), and by amino acid analysis at the Hospital for Sick Children (Toronto, Canada) [40].
iMALDI reagents
Goat polyclonal anti-angiotensin antibody (pAb) (SC-7419) was purchased from Santa Cruz Biotechnology. Magnetic protein G Dynabeads were purchased from Invitrogen. Synthetic peptides (natural and SIS) were synthesized in house [40]. TFA was purchased from Fisher Scientific. PBS, CHAPS, EDTA, maleic acid, neomycin trisulphate salt hydrate, PMSF, ammonium bicarbonate, α-cyano-4-hydroxycinnamic acid (CHCA), ammonium citrate dibasic, LC/MS-grade H2O, and LC/MS-Grade ACN were purchased from Sigma Aldrich.
RIA procedure
Preparation of reference standards
Reference standards were prepared by dissolving purified Ang-I in a buffer containing 1% BSA in 0.1 M Tris, to make a stock concentration of 5000 μg/mL Ang-I. The stock solution was spiked and then serially diluted to make a calibration curve at 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 ng/mL in a 1% BSA in 0.1 M Tris buffer.
Generation of angiotensin I
Frozen plasma samples were thawed for 5 minutes in a RT water bath followed immediately by immersion in an ice-water bath, where they remained, except for the 37°C generation step. Patient samples, reference standards, and controls were aliquoted in duplicate for the 37°C incubation and the blank (ice-water bath) incubation. Ten μL of rabbit anti-angiotensin in a 1 M Tris/0.2 M EDTA buffer at pH = 7.4 were added to each conical polystyrene tube (Evergreen Scientific), followed by 50 μL of standards and unknowns. The 37°C-incubation tubes were placed in a 37°C water bath for exactly 1 h; the blank tubes were kept in the ice bath. At the end of the incubation period, the 37°C samples were immediately returned to the ice bath.
RIA analysis
The 125I-Ang-I tracer was diluted in 0.1 M Tris to yield 18,000-22,000 CPM/mL, and 1 mL was added to each tube. Fifty μL of plasma with undetectable PRA was added to all standard tubes to act as a suitable matrix. Tubes were vortexed and incubated at 4°C for 48–72 h to allow the competitive binding of the 125I-Ang-I to the anti-angiotensin antibody.At the end of incubation, 500 μL of cold dextran-coated charcoal in 0.1 M Tris was added to each sample, mixed, and centrifuged. The supernatant was decanted into round-bottom polystyrene tubes (Simport). The supernatants were counted on a Wallac 1260 MULTIGAMMA II gamma counter (Perkin Elmer). Data reduction was performed using StatLIA Enterprise 3.2 (Brendan Technologies). PRA was calculated as ng/L/s using blank subtraction (Equation 1).
(1)
LC-MS/MS procedure
Preparation of reference standards
Reference standards were prepared in the same manner as the RIA analysis. Concentrations of 100, 30, 9.0, 2.7, 1.35, 0.675, and 0.3375 ng/mL were prepared in a 1% BSA in 0.1 M Tris buffer.
Generation of angiotensin I
Plasma samples were received for LC/ESI-MS/MS analysis in an ice bath, immediately after being aliquoted for RIA analysis. Two hundred and fifty μL of reference standards and plasma samples were added in duplicate to two square polypropylene 2-mL 96 well plates (Corning Inc.), for the 37°C and blank determinations, respectively. The 37°C generation plate contained 50 μL of the generation buffer (1 M Tris, 0.2 M EDTA, and 1 mM PMSF at pH 5.5) resulting in a plasma pH of 6.0. The 37°C generation plate was mixed briefly, and placed in a 37°C water bath for exactly 3 h. The blank plate was extracted by SPE immediately after samples were aliquoted. The blank-subtraction plate was not subjected to a 3-h incubation on ice.
Extraction of angiotensin I
Three hundred μL of Ang-I-SIS internal standard, at 10 ng/mL in 10% FA, was aliquoted to each sample, and mixed briefly. A Strata-X 33 μ polymeric reversed phase 60 mg 96 well plate (Phenomenex) was conditioned with 1 mL of methanol followed by 1 mL of 5% FA with vacuum applied at 200 mbar for 1 min for each condition step. The entire sample (550 μL for blank samples and 600 μL for 37°C samples) was loaded onto the plate and flowed through using vacuum applied at 200 mbar for 1 min. The plate was washed with 5% FA followed by 20% methanol with vacuum applied at 200 mbar for 1 min for each wash step and dried under 200 mbar of vacuum for 10 min. Ang-I was eluted from the plate with 250 μL of methanol and collected in a 2-mL polypropylene round-bottom 96 well plate (NUNC/Thermo Fisher) with 200 mbar of vacuum applied for 2 min.
LC/ESI-MS/MS analysis
LC/ESI-MS/MS analysis was performed on a UFLC 20 AC (Shimadzu Corporation) with an AB SCIEX API5000 triple-quadrupole (Applied Biosystems). Twenty microliters of sample extracts were injected onto a 50 mm x 2 mm Jupiter 4 μ Proteo 90A Analytical Column (Phenomenex). Mobile phases A and B were 0.2% FA in water, and 0.2% FA in methanol, respectively. Using a 0.5 mL/min flow rate, Ang-I was eluted at 95% B after 0.5 min of column conditioning at 10% B and a gradient to 95% B in 1 min. The system was returned to starting conditions in 0.1 min and re-equilibrated at 10% B for 2.4 min. The total cycle time was 6 min per sample. Triply-charged ions 433.2→647.5 (quantifier) and 433.2→619.3 (qualifier) were monitored for Ang-I and 436.6→657.5 for the SIS-Ang-I internal standard. Calibration curves were fit with 1/x2 linear regression. PRA was calculated in ng/L/s using Equation 1.
iMALDI procedure
Washing of protein G dynabeads
Protein G Dynabeads were used at a ratio of 5 μL of bead slurry (30 mg/mL) per 1 μg of pAb. Beads were washed 7 times with 1 mL of 25% ACN/1xPBS/0.03% CHAPS, and 3 times with 1xPBS/0.03% CHAPS solution in a 1.5 mL Maxymum Recovery tube (Axygen Scientific). Beads were pelleted with a Dynamag-2 (Invitrogen), allowing 20 s to pellet the beads before removing the supernatant.
Conjugation of protein G dynabeads and pAb
Antibody was added to Protein G Dynabeads based on experimental need (1 plate used roughly 23 μg of pAb) at a ratio of 1 μg of pAb to 5 μL of washed beads. A solution of 1xPBS/0.03% CHAPS was added to give a final volume of 7 μL of liquid per 1 μg of pAb. The sample was quickly spun to collect beads at the bottom of the tube and then lightly vortexed to resuspend them. Beads were placed on a Labquake rotor (Fisher Scientific) for 1 h at RT with end-over-end rotation. Following conjugation, beads were pelleted and washed 5 times with 1 mL of 1×PBS/0.03% CHAPS. Once washed, beads were resuspended in 1×PBS/0.03% CHAPS to a pAb concentration of 0.01 μg/μL. Twenty μL of bead solution was transferred to each well of a 96-well skirted PCR plate, which results in 0.2 μg of pAb per capture well.
Generation of angiotensin I
Plasma samples were thawed in a RT water bath for 5 min and then placed on ice. Before use, plasma was vortexed and a 200 μL aliquot was added to a 1.5-mL Maxymum Recovery tube, to give a final concentration of 5 mM EDTA, 25 mM maleic acid, 275 μM neomycin trisulphate, and 1 mM PMSF. The final sample pH was 6. The total volume of all inhibitors added was 12.23 μL, resulting in a 6% dilution. Samples were vortexed and split into two tubes containing 100 μL each, representing the blank and generated sample. The blank sample was placed on ice, and the generated sample was placed in a 37°C thermoshake rotor (Eppendorf), shaking at 1000 rpm for exactly 1 h.
Antigen capture
Beads were pelleted in the 96 well PCR plate (Axygen) by placing it on a Dynal MPC-96S plate magnet and the supernatant was removed. Tubes were continually vortexed during Ang-I generation, and were then placed on ice. Triplicate analyses of patient blank and generated samples were added to the PCR plate (6 captures per patient). This was repeated for each patient sample, with a total of 13 patients analyzed per plate (78 wells). Triplicate six-point calibration curves at concentrations of 14.8, 7.4, 3.7, 1.9, 0.9, and 0 ng/mL natural Ang-I were prepared in 35 μL of 1×PBS/0.03% CHAPS. Both reference standards and patient samples were spiked with SIS Ang-I internal standard to a concentration of 4.6 ng/mL. After addition of the standards, samples were mixed by pipetting with an 8-channel pipette (Fisher Scientific). The PCR plate was sealed with an Axymat (Axygen), taped to a Labquake rotor, and incubated at 4°C for 1 h with end-over-end rotation.
Bead washing and MALDI spotting
Following 1 h of antibody capture, the plate was removed and placed on the PCR plate magnet. Using a multichannel pipette, samples were washed column by column, starting with column 1. The capture solution (plasma and standards) was removed from the pelleted beads. The PCR plate was removed from the magnet and 100 μL of 15% ACN/25 mM ammonium bicarbonate was used to resuspend the beads. The PCR plate was placed back on the magnet and the beads were allowed to pellet for 20 s. This was repeated an additional 2 times for a total of 3 washes. After the final wash, all buffer was removed and the plate was removed from the magnet. Beads were resuspended in 3 μL of 15%ACN/25 mM ammonium bicarbonate, and the entire bead solution was spotted on a MALDI target (part number: 4352802, AB SCIEX). The procedure was performed for each column separately to ensure that the time from first wash to spotting on the MALDI target was consistent across all samples. After all wells had been spotted, the plate was left at RT until all spots were dry and uniform in appearance. Two μL of MALDI matrix solution (containing 3 mg/mL CHCA, 1.8 mg/mL ammonium citrate, 70% ACN, and 0.1% TFA) was overspotted onto each bead spot to elute the peptide and prepare the spot for MALDI analysis.
MALDI analysis
Samples were analyzed on an AB SCIEX 4800 MALDI TOF-TOF using Reflector Positive mode, from m/z 800 to 4000. Samples were analyzed in batch mode, after checking before acquisition to ensure that the laser setting produced sufficient intensities for both blank and generated sample. A total of 1250 laser shots were acquired for each spot (10 sub-spectra with 125 shots per sub-spectrum) using a fixed laser intensity. Representative spectra from a 4°C and 37°C capture are shown in Figure 4. Figure 4 also shows a peak at m/z 1295.68 – 1 Da lower than the Natural Ang – which was associated with the antibody. The blank subtraction method for PRA determination removes any potential interference from the 13C peak of this component (see Additional file 1).
Data analysis and PRA determination
Data was analyzed using AB SCIEX’s Data Explorer software. Peak heights were recorded after performing a baseline correction. Two peak heights were recorded -- those of Natural Ang-I (m/z 1296.68) and SIS Ang-I (m/z 1306.68), and the relative response (RR) was reported as the Nat:SIS ratio. These RR values were compared to the calibration curve. PRA was calculated in ng/L/s by the blank subtraction method (Equation 1). Regressions were performed by the method of Passing and Bablok using cp-R, a graphical user interface to the R statistical programming language [41].
