Biochimica et Biophysica Acta 1854 (2015) 547–558

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An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI)☆ Robert Popp a, David Malmström a, Andrew G. Chambers a, David Lin a, Alexander G. Camenzind a, J. Grace van der Gugten b, Daniel T. Holmes b, Michael Pugia c, Marta Jaremek d, Shannon Cornett e, Detlev Suckau f, Christoph H. Borchers a,g,⁎ a

University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria-British Columbia V8Z 7X8, Canada University of British Columbia, Department of Pathology and Laboratory Medicine, St. Paul's Hospital, 1081 Burrard Street, Vancouver, British Columbia V6Z 1Y6, Canada c Siemens Healthcare Diagnostics, Elkhart, IN, USA d Siemens Healthcare, Erlangen, Germany e Bruker Daltonics, Billerica, MA, USA f Bruker Daltonik, Bremen, Germany g Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada b

a r t i c l e

i n f o

Article history: Received 1 July 2014 Received in revised form 26 September 2014 Accepted 9 October 2014 Available online 16 October 2014 Keywords: Hypertension Angiotensin-I Plasma renin activity (PRA) Matrix assisted laser desorption/ionization (MALDI) mass spectrometry Immuno-MALDI (iMALDI) Automation

a b s t r a c t Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC–MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC–MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7 h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC–MS/MS. The assay is performed on 350 μL of plasma, and has a linear range from 0.08 to 5.3 ng/L/s in the reflector mode, and 0.04 to 5.3 ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC–MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R2 value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Approximately 1 billion people worldwide suffered from hypertension in 2008 [1]. Primary aldosteronism, a form of secondary hypertension, accounts for an estimated 10% of all hypertensive subjects [2]. Compared with patients suffering from primary hypertension, patients with primary aldosteronism have been shown to be at a higher risk of cardiovascular events, such as stroke, myocardial infarction or atrial fibrillation [3,4]. Furthermore, once considered a rare disease, primary aldosteronism is now considered to be the most common form of hypertension

☆ This article is part of a Special Issue entitled: Medical Proteomics. ⁎ Corresponding author at: University of Victoria-Genome BC Proteomics Centre, 31014464 Markham St, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada. Tel.: +1 250 483 3221; fax: +1 250 483 3238. E-mail address: [email protected] (C.H. Borchers).

http://dx.doi.org/10.1016/j.bbapap.2014.10.008 1570-9639/© 2014 Elsevier B.V. All rights reserved.

that is curable by specific treatment [5]. Thus, the estimated 7.5 million hypertension-related deaths annually reflect the importance of effective diagnosis of this condition [1]. Primary aldosteronism is caused by a dysregulation of the renin– angiotensin–aldosteronesystem (RAAS) — a coordinated enzymatic cascade of the circulatory system that regulates arterial pressure, tissue perfusion, electrolyte balance, and extracellular volume [6]. The proteolytic enzyme renin, mainly secreted by the kidneys, cleaves the glycoprotein angiotensinogen (released into the circulation by the liver) to angiotensin I (Ang I) [7]. Ang I is a decapeptide (DRVYIHPFHL) with no direct physiological activity. Particularly in the lung, Ang I is converted to the octapeptide Ang II (DRVYIHPF) by the cleavage of two Cterminal amino acids. This hydrolysis reaction is catalyzed by the angiotensin-converting enzyme (ACE) [8]. Among other effects, Ang II causes vasoconstriction, mainly mediated via Ang II type 1 (AT1) receptors, which leads to increased release of aldosterone by acting on the

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adrenal cortex [9]. Aldosterone is a mineralocorticoid hormone that is key to the regulation of the sodium and potassium balance, and therefore for the balance of circulating blood volume and blood pressure. Increased Ang II levels results in elevated aldosterone production, which leads to a negative feedback, thereby inhibiting renin secretion [10]. Patients suffering from primary aldosteronism show elevated levels of circulating aldosterone as well as suppressed plasma renin activity (PRA) [11]. This increase in circulating aldosterone is independent of the Ang II plasma concentration and is mainly caused by bilateral adrenal hyperplasia (65–70% of cases) or an aldosterone-producing adenoma (30–35% of cases) [2,12]. Additional, but less frequent, causes of primary aldosteronism include unilateral adrenal hyperplasia and adrenocortical carcinoma [13]. The process from diagnosis to treatment of primary aldosteronism consists of four steps: screening, confirmatory testing, subtypeclassification, and treatment. Screening for primary aldosteronism is typically performed by analyzing a patient's blood sample to determine the aldosterone–renin ratio (ARR) [14] — a screening test which was introduced in the 1980s [15]. The ARR compares the aldosterone concentration with active plasma renin, either determined as plasma renin activity (PRA), or as the direct renin concentration (DRC) [16]. Both methods of determining plasma renin are used clinically, and give complementary, but different information. For example, if patients are receiving renin inhibitor therapy, this can lead to inhibited renin activity, but the direct renin concentration measurement is not affected [7]. Elevated ARR results indicate autonomous aldosterone release and are an indication of primary aldosteronism. In addition to elevated Ang II or the other main causes for autonomous aldosterone secretion (bilateral adrenal hyperplasia and aldosterone-producing adenoma), aldosterone secretion can also be triggered by low plasma Na+ or high plasma K+ concentrations (hyperkalemia), adrenocorticotropic hormone (ACTH), or other stimuli [10]. These factors can lead to elevated ARR-results that are not related to primary aldosteronism, thereby leading to false-positive results. Thus, confirmatory aldosterone suppression testing is usually performed to confirm or reject the diagnosis of primary aldosteronism [17]. PRA determination by radioimmunoassay (RIA) was introduced in the 1960s [18], further developed throughout the 1970s [19,20], and is still the most commonly used method today [14]. Statistical evaluation of 4170 untreated participants in a hypertension treatment program suggested that PRA values fall into three ranges: low PRA (b 0.18 ng/L/s), medium PRA (b0.18–1.25 ng/L/s), and high PRA (N1.25 ng/L/s) [21]. Like all immunoassays, RIAs suffer from the possibility of cross-reactivity and therefore the possibility of falsely-elevated PRA results. Additionally, RIAs make use of radioactive assay components which not only require laboratories with a special license to handle radioactive materials, but also puts the personnel performing the assay at risk. Furthermore, RIAs have a limited dynamic range, and, depending on the incubation conditions, can be time-consuming (ranges of 3 h total assay time [20] to approximately 72 h [22] have been reported in the literature). In addition to the well-established immunoassays for PRA determination, MS-based methods have recently been developed, including liquid chromatography–mass spectrometry (LC–MS) methods that typically involve immuno-capture of Ang I [23], or solid phase extraction [24]. These approaches have the advantage of being highly specific due to the assignment of the target compound by mass, thereby eliminating cross-reactivity as a source of error, but they suffer from relatively low sample throughput and the increased cost and complexity of LC– MS instrumentation [25]. A relatively new approach to overcome the disadvantages with current protein/peptide quantitation techniques is immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) [26–30]. This technique is based on immuno-enrichment of endogenous target peptides and stable isotope-labeled standard analogs (SIS peptides) on affinity-beads that carry immobilized antibodies against the target peptides. The bead–antibody–peptide conjugate is washed and spotted directly onto

a MALDI target without prior elution. Application of the acidic MALDI matrix solution elutes the captured target endogenous and SIS peptides, which then co-crystallize with the matrix molecules. The samples are then analyzed by MALDI. The SIS peptides are used as internal standards and allow absolute quantitation of the target peptide. The iMALDI approach is highly specific and sensitive, and allows analyte confirmation by MS/MS with TOF/TOF instruments. Furthermore sample losses are kept to a minimum since the beads with bound analyte are directly spotted onto the MALDI target. This eliminates adsorption of the analyte by the walls of the Eppendorf tube which can occur if the analyte is in solution. Also, the iMALDI technology has the capability for being automated, thereby constituting a method that has the potential to replace current clinical protein quantitation techniques. We previously developed an immuno-MALDI (iMALDI) approach for PRA determination which utilizes anti-Ang I antibodies immobilized to affinity beads. The antibody beads simultaneously capture endogenous Ang I from plasma along with the stable isotope-labeled Ang I (Ang I SIS) for absolute quantitation. Each plasma sample is split and incubated either at 37 °C for 3 h (which allows Ang I generation due to the enzymatic cleavage of angiotensinogen by renin), or on ice (which prevents this reaction), followed by a one-hour affinity-capture period. Determining the difference in Ang I concentration for the two plasma incubation conditions allows the calculation of the patient's plasma renin activity. Comparison of this iMALDI approach to RIA and LC–MS/MS methods for 64 patient samples indicated a strong correlation (R2 N 0.94) [22]. To increase sample throughput, reduce assay variability between samples, and to decrease the likelihood of errors being made by laboratory personnel during sample preparation, we have now automated the liquid handling steps of the iMALDI PRA assay sample preparation on an Agilent Bravo, followed by analysis on a Bruker Microflex LRF MALDITOF instrument, with the final goal of generating an assay suitable for the clinical laboratory. In recent years, MALDI-TOF instruments from several vendors, including Shimadzu's AXIMA system [31], BioMérieux's VITEK MS [32], and Bruker's BioTyper CA [33], have been introduced into clinical laboratories for microbial identification, and offer a fast and inexpensive technology with the potential to replace or supplement conventional phenotypic identification. Bruker's BioTyper CA system, which is a linear mode model of the Microflex, received U.S. FDA clearance in November 2013, and has already been sold or leased to more than 1000 laboratories worldwide [34]. In this current paper, we show that these MALDITOF instruments, which are already in place in clinical laboratories, have the potential to be used for robust and high-throughput PRA determination. 2. Material and methods 2.1. Plasma samples Healthy control human plasma was obtained from Bioreclamation (K2 EDTA plasma, Cat-#: HMPLEDTA2, pooled from 5 males and 5 females). Human patient samples were selected from the routine primary aldosteronism screening program at St. Paul's Hospital, Vancouver. Ethics approval was granted by the ethics board of St. Paul's Hospital and the University of British Columbia. The patient samples were collected into pre-chilled EDTA tubes, centrifuged at 4 °C, and frozen at − 20 °C for a maximum of ten days. Specimens were thawed for 5 min in a room temperature water bath and kept at 4 °C until LC– MS/MS analysis. Samples were then refrozen and maintained at -80 °C until analyzed by iMALDI at the University of Victoria (UVic)-Genome BC Proteomics Centre. 2.2. LC–MS/MS reagents and procedure The LC–MS/MS procedure for PRA determination and the reagents used have been published previously [22].

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2.3. iMALDI reagents Ammonium bicarbonate (AmBic), ammonium citrate dibasic, CHAPS (≥98%), albumin from chicken egg white (N 98%), ethylenediaminetetraacetic acid (EDTA), alpha-cyano-4-hydroxycinnamic acid (HCCA), phosphate buffered saline (PBS), and phenylmethanesulfonyl fluoride (PMSF) were obtained from Sigma Aldrich. Trifluoroacetic acid (TFA) was obtained from Thermo Fisher Scientific. LC–MS grade acetonitrile (ACN), H2O, methanol and acetic acid were purchased from Fluka. Tris(hydroxymethyl)aminomethane (Tris base) was purchased from Roche Diagnostics. Peptide Calibration Standard II was purchased from Bruker. Dynabeads Protein G magnetic beads (2.8 μm; 30 mg/mL) were purchased from Life Technologies™. A commercially-available anti-Ang I goat polyclonal antibody (sc-7419) was purchased from Santa Cruz Biotechnology. Synthetic Ang I (Ang I Nat, DRVYIHPFHL) and stable isotope labeled Ang I (Ang I SIS, DRVYIHPFHL) were synthesized at the University of Victoria — Genome BC Proteomics Centre (Victoria, BC, Canada) by using solid phase peptide synthesis (SPPS), as previously described [35]. After synthesis, the lyophilized peptides were resuspended in 30% ACN/ 0.1% formic acid (FA), and stored as stock solutions at −80 °C until used. The heavy Ang I SIS peptide differs from the natural form of Ang I (Ang I Nat) peptide by 10 Da due to the incorporation of a stable isotope-coded arginine residue (13C, 15N). The wash buffer 1×PBS + 0.015% CHAPS (PBSC) was freshly prepared every 2–3 days and stored at 4 °C. The wash buffers (25% ACN/ PBSC and 25 mM AmBic) were prepared fresh every day. The 25 mM AmBic buffer was stored protected from light until used. Prior to being used, all wash buffers were brought to room temperature. Calibration standards for a six-point calibration curve were prepared manually by diluting Ang I Nat and Ang I SIS stock solutions with PBSC to varying Ang I Nat (0, 5, 10, 20, 40, and 80 fmol/μL) and constant Ang I SIS concentrations (25 fmol/μL, calibration standards F, E, D, C, B and A, respectively). The standards were stored at − 20 °C until used. On the day of experiment, the standards were thawed on ice, transferred manually to a 96-well PCR plate (bead standard plate, 35 μL per well), and kept on ice until used. The Ang I generation buffer was prepared from a 1 M Tris/0.2 mM EDTA aqueous buffer (adjusted to pH 5.5 with acetic acid) and a PMSF solution (100 mM in methanol). Both solutions were stored up to 1 month at 4 °C. On the day of experiment, the two solutions were mixed to yield an Ang I generation buffer which contained 1 M Tris, 0.2 mM EDTA, and 1 mM PMSF. The Ang I generation buffer was then transferred manually to a 96-well PCR plate (reagent plate, 120 μL per well), and stored at 4 °C until used. HCCA-matrix (containing 3 mg/mL CHCA, 1.8 mg/mL ammonium citrate, 70% ACN, and 0.1% TFA) was prepared fresh every 3 days, and stored at room temperature and protected from light until used [36]. Prior to the automated matrix application step, the matrix was transferred manually to a 96-well PCR plate (matrix plate, 140 μL per well). Chicken albumin from egg white (CEWA)/1×PBS buffer (3 mg/mL) was prepared fresh every day and stored at 4 °C until used. 2.4. Automated iMALDI PRA procedure An Agilent Bravo robotic workstation, equipped with a gripper designed to grip the 96-well plates and a 96-channel LT head, was used for automated liquid handling in a 96-well format. Four protocols were created which automatically perform plasma preparation tasks, bead and standard transfers, bead washing and spotting, and matrix spotting. The Bravo platepad setup and the layouts for the four protocols are shown in the Supplementary Information, Figs. S1–5. The following describes the overall workflow for PRA determination of 29 patient samples, including the manual and automated steps in the iMALDI PRA procedure (Fig. 1). Since the overall procedure is not completely automated (e.g., 96-well plates are transferred manually to

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an incubator) the terms “automated assay”, “automated sample preparation”, or “automated procedure” used throughout the paper refer to the assay, sample preparation, or the portion of the procedure that includes the automated liquid handling steps. 2.4.1. Plasma preparation To prevent significant Ang I generation, plasma samples were thawed in a water bath at room temperature for only 5 min and were then immediately placed on ice for the remainder of the thawing process. For each sample, 350 μL of thawed plasma were transferred manually to one well of an Axygen 1.1-mL deep well plate (plasma plate). The plate was then centrifuged for 10 min at 0 °C and 3000 × g in a Beckman Coulter Allegra X-22R Centrifuge. Next, 350 μL of CEWA/ 1×PBS buffer was transferred manually into three wells (A1, A2, and A3) of the plasma plate. Before starting the “plasma preparation” protocol on the Agilent Bravo, the standard platepad on position 5 was exchanged for another standard platepad that had been stored at −20 °C for at least 30 min. The Bravo was then used to automatically mix 250 μL of plasma supernatant or 250 μL of CEWA/1 × PBS with 50 μL of Ang I generation buffer, after which the resulting solution was pipetted automatically into two 96-well PCR plates (4 °C and 37 °C incubation plates, 3 replicates per plate, 34 μL per replicate). Each replicate contained 28.3 μL of plasma and 5.7 μL of Ang I generation buffer. 2.4.2. Angiotensin I generation The 4 °C incubation plate was stored on ice, and the 37 °C incubation plate was incubated for 3 h at 37 °C and 1000 rpm on an Eppendorf Thermomixer R to allow generation of Ang I due to endogenous renin activity. 2.4.3. Conjugation of anti-Ang I antibodies to magnetic Protein G Dynabeads Conjugation of anti-Ang I antibodies to the magnetic beads was performed manually during the 3-hour Ang I generation period, on each day of the experiment. The bead slurry (217 μL for 29 patient samples) was transferred to a 15 mL Falcon tube and washed seven times with 25% ACN/PBSC solution, and three times with PBSC (6.5 mL per wash). A 15 mL EMD Millipore PureProteome magnetic stand was used to pellet the beads for ~30–45 s during each washing step. The CHAPS in the wash buffer prevents the beads from sticking to the walls, which significantly improves the manual washing and subsequent automated liquid handling performance. After the last wash, the beads were transferred to a 0.6 mL MAXYMum Recovery microcentrifuge tube (Axygen), and, after pelleting the beads with an Invitrogen DynaMag-2 magnet, as much of the liquid was removed as possible. Then, 217 μL of PBSC and 217 μL of sc-7419 antibody (43.4 μg) were added to the beads, the tube was vortexed, and the beads were incubated with the solution at 4 °C for 1 h while rotating on a Thermo Scientific Labquake Tube Rotator at 8 rpm. Note that the transfer from the 15 mL Falcon tube to the 0.6 mL microcentrifuge tube was performed to prevent the bead–antibody solution from flowing into the lid of the Falcon tube while rotating, which would require an additional spin-down with a larger centrifuge. The 0.6 mL microcentrifuge tube could be spun down with a benchtop microcentrifuge instead. After the 1-hour incubation, the bead–antibody conjugate was transferred to a 15 mL Falcon tube, washed three times with PBSC (6.5 mL per wash), and resuspended in 2604 μL PBSC. The resuspended bead–antibody conjugate was then aliquoted manually into the bead standard plate (106 μL per well), and the plate was rotated at 4 °C on a Thermo Scientific Labquake Tube Rotator at 8 rpm until used. 2.4.4. Angiotensin I capture After the 3-hour Ang I generation period, the 37 °C incubation plate was stored on ice for 10 min to stop the generation of Ang I. The standard platepad on position 5 was then exchanged for another standard platepad that had been stored at − 20 °C for at least 30 min. The “bead and standard transfer” protocol of the Agilent Bravo was then

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Fig. 1. Workflow of the iMALDI procedure for PRA determination of 29 patient samples, including the manual and automated steps.

used to transfer 5 μL of calibration standards A–F or the SIS standard (calibration standard F), and 12 μL of resuspended bead–antibody conjugate from the bead standard plate. In this way, the incubated plasma is mixed with the SIS standard (calibration standard F), and the CEWA/ 1×PBS wells are mixed with the calibration standards A–F. After mixing, the bead–antibody conjugate is added to the 4 °C and 37 °C incubation plates for affinity capture. The two incubation plates were then incubated for 1 h at 4 °C while rotating on a Thermo Scientific Labquake Tube Rotator at 8 rpm.

2.4.5. Bead washing and spotting After the 1-hour Ang I capture period, the “bead washing and spotting”-protocol was used to automatically wash and spot the bead–antibody–peptide conjugate with the Agilent Bravo, starting with the 4 °C incubation plate. Each well was washed three times with 70 μL of 25 mM AmBic. After the last wash, the beads were resuspended in 7 μL of 25 mM AmBic per well, and the entire volume was spotted onto four Bruker MSP BigAnchor 96 MALDI targets.

For pelleting the bead–antibody–peptide conjugates for the first two washes, a DynaMag-96 side skirted magnet was used to pull the beads to the side of the wells. This allows removal of almost all of the liquid from the wells. For removing the wash buffer after the third wash, a VP Scientific 771RM-1 magnet was used to pull the beads to the bottom of the well. This allows removal of all but 7 μL of the liquid. The 7 μL that was left is required to fully resuspend the beads prior to spotting. Removing the entire incubation liquid and the wash buffer after the first two washes was crucial to preventing introduction of air bubbles during the third wash, which would have led to unequal volumes of leftover wash buffer being required for resuspending the beads. Because the Bruker MSP targets are only ¼ the size of a standard microtiter plate, a custom adapter was built to arrange the four targets into a microtiter-plate format. Each MSP BigAnchor target contains 96 spots, thereby resulting in an overall 384-well format for the four targets combined, which allowed spotting of the 4 °C incubation plate samples, and subsequent offset spotting of the 37 °C incubation plate samples (Fig. 2). Additionally, four spots of 0.5 μL of Bruker peptide

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calibration standard II were spotted manually in each quadrant of each MSP MALDI target for calibrating the Microflex. A small USB-powered fan was used to accelerate the drying process. 2.4.6. Matrix application After the spots were dried, the “matrix application”-protocol on the Agilent Bravo was used to automatically transfer 1 μL of HCCA-matrix from the matrix plate to each sample spot. The acidic pH of the matrix solution elutes the peptides from the antibodies and allows cocrystallization with the HCCA-matrix. Additionally, 1 μL of HCCAmatrix was transferred manually to the dried calibration spots. 2.4.7. MALDI analysis The sample spots were analyzed on a Bruker Microflex LRF instrument. For each spot, one spectrum was acquired in the positive reflector and positive linear mode (1000 shots, fixed laser intensity, m/z 500 to 4000, ion suppression up to 700 Da, 15 shots at raster spot). AutoXecute methods for both measurement modes were written in the Bruker FlexControl 3.3 software to automatically acquire data from the MALDI targets. Methods for both measurement modes already included in the Bruker FlexAnalysis 3.4 software were modified to automatically perform internal calibration, smoothing, and baseline subtraction. 2.4.8. Data analysis and PRA calculation In FlexAnalysis 3.4, each spectrum was analyzed for the contaminant signal (m/z 1295.7), Ang I Nat (m/z 1296.7) and Ang I SIS (m/z 1306.7) peak intensities. The contaminant signal had previously been traced back to the antibody (probably from the antibody purification step) and MS/MS data indicated that it was a modified Ang I which is amidated at the C-terminus [22]. The contaminant 2-13C isotope peak overlaps with the Ang I Nat 12C isotope peak (m/z 1296.7). The contaminant's isotopic pattern was determined in each experiment by performing buffer captures in triplicate without Ang I Nat added. This allowed the calculation of a correction factor, which subtracts the influence of the contaminant on the Ang I Nat peak (m/z 1296.7) in each patient sample and calibration spot. The “adjusted” Ang I Nat signal intensity, and the original Ang I SIS peak intensity were used to calculate the relative response ratio (RR, Nat/SIS intensity ratio). By comparing the Nat/SIS ratios to the calibration curve, the Ang I concentrations for each plasma sample were determined (Fig. 3). The final Nat/SIS ratios of each patient sample (incubated at 4 °C and 37 °C) were calculated

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from the average of the triplicate captures per incubation temperature. The PRA calculation was performed according to Eq. (1). PRA ¼ ½Ang I37



C −½Ang


Iice =Δt Ang

I generation

ð1Þ

2.5. Linear range To determine the linear range, limit of detection (LOD), and lower limit of quantification (LLOQ) of the automated iMALDI PRA assay, Ang I standards were made by serial dilution of the Ang I SIS stock solution with PBSC with the addition of a constant amount of Ang I Nat. Following the procedure described in the automated iMALDI PRA procedure, each standard concentration was spiked into healthy control human plasma (n = 3). The final amounts per sample spot were 4.9, 9.8, 19.5, 39.1, 78.1, 312.5, 625, 1250, and 2500 fmol Ang I SIS, and 125 fmol Ang I Nat. 2.6. iMALDI PRA precision testing For the determination of the precision of the iMALDI PRA assay, patient samples with known PRA values, as determined by LC–MS/MS at St. Paul's Hospital and described previously [22], were grouped into low, medium, and high PRA values, following the PRA ranges defined by Alderman et al. [21]. The patient samples were thawed for 5 min in a room temperature water bath, placed on ice, and combined into three PRA pools. Aliquots of each pool were stored at −80 °C until the day of analysis. Each aliquot was then treated as a separate patient sample, using the automated iMALDI PRA procedure. Intraday precision was tested on five replicates of each PRA pool on one day; interday precision was tested on one replicate per PRA pool on five consecutive days. 2.7. Comparison of manual and automated sample preparation To compare the automated sample preparation procedure with the completely manual sample preparation for PRA determination, 13 aliquots of the same healthy control human plasma and a calibration curve were prepared. The analysis was performed once using the automated iMALDI PRA procedure and the Agilent Bravo for sample preparation, and once using the same protocol, but preparing the automated steps manually. The calibration curve and the 13 samples represent

Fig. 2. MALDI target layout for 29 patient samples and one calibration curve (192 captures), consisting of four Bruker MSP BigAnchor MALDI targets arranged on a custom-built adapter to produce a 384-well format.

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Fig. 3. PRA is calculated by determining the amount of Ang I generated during the 3-hour Ang I generation period.

half of the capacity of the assay (96 captures), as compared to the full assay capacity (192 captures) which corresponds to one calibration curve and 29 samples (see Fig. 1). To speed up the manual sample preparation, a multichannel pipette was used for mixing the plasma with generation buffer and aliquoting it into the 4 °C and 37 °C plasma plates, transferring the beads, and the washing steps. For matrix spotting, a single-channel pipette was used.

2.8. Method comparison: PRA determination by iMALDI vs. LC–MS/MS for 188 patient samples To evaluate the suitability of the iMALDI assay for the clinical measurement of PRA, the PRA values of 188 patient samples were determined by iMALDI using the automated iMALDI procedure, and compared to the PRA values obtained with the LC–MS/MS method currently used for routine clinical screening for primary aldosteronism at St. Paul's Hospital, Vancouver.

3. Results and discussion

The LLOQ was defined as the lowest concentration at which the variability of the replicates was b20%, with an intensity of at least 5 times higher than the blank. The criteria for determining the linear range were the following: the percent-difference of the SIS/Nat ratio to the regression line must be within ± 20% at the LLOQ, and ± 15% for the higher concentrations of Ang I, and must show a precision within ±20% for the triplicate captures at each point of the calibration curve. Using these criteria, the linear range of the iMALDI PRA assay was determined to be 19.5–1250 fmol (0.08–5.3 ng/L/s) in the reflector mode, and 9.8 fmol to 1250 fmol (0.04–5.3 ng/L/s) in the linear mode (Fig. 4). The linear mode therefore extended the linear range to Ang I levels that were a factor of 2 lower than the reflector mode. Furthermore the slopes of both curves were very similar with 0.0068 and 0.0072, indicating nearly identical response ratios for both measurement modes. The precision of the linear mode was lower than in the reflector mode. The average %CV of the three replicates for all Ang I SIS concentrations within the linear range was 1.9% in the reflector mode and 5.5% in the linear mode. Since the same sample spots were analyzed in both the reflector and linear modes, the higher imprecision of the linear mode can be explained by the nature of the linear mode, which has lower resolution and lower S/N levels than the reflector mode. This observation is consistent with findings by Anderson et al. [37], who investigated the precision of heavy-light peptide ratios as determined by MALDI-TOF MS. The linear range of the iMALDI PRA assay in both the reflector and linear modes is suitable for accurately distinguishing between low (b0.18 ng/L/s), medium (0.18–1.25 ng/L/s) and high (N1.25 ng/L/s) PRA patient samples (ranges defined by Alderman et al. [21]) and therefore covers the clinically relevant range of PRA samples. Furthermore, the sensitivity of the iMALDI assay in both the reflector and linear mode is suitable for clinical utility and is comparable to published LC–MS/MS methods for determining PRA, e.g., by Carter et al. (LLOQ = 0.04 ng/L/s with a 6.5-hour Ang I generation period) [24]. The sensitivity of the iMALDI assay is in agreement with the Endocrine Society's Clinical Practice Guidelines, which recommend a minimum sensitivity of 0.6–0.08 ng/L/s for clinically suitable PRA assays [14], and is below the established PRA cut-off value of 0.18 ng/L/s for low PRA patients [38].

3.1. Linear range 3.2. iMALDI PRA precision testing The limit of detection (LOD) of the assay was defined as the lowest concentration with a signal-to-noise (S/N) of at least 3. The LOD was 4.9 fmol (0.02 ng/L/s) in the reflector mode and 9.8 fmol (0.04 ng/L/s) in the linear mode. Each of the corresponding LOD concentrations of Ang I SIS (m/z 1306.7) showed a S/N of ≥5.

To test the intraday precision of the iMALDI PRA assay, five replicates of low, medium, and high PRA sample pools were analyzed on a single day. The interday precision was determined by analyzing one replicate of the low, medium, and high PRA sample pools across five days. The

Fig. 4. The linear range of the iMALDI PRA assay is 19.5–1250 fmol (0.08–5.3 ng/L/s) in the reflector mode (A) and 9.8 fmol–1250 fmol (0.04–5.3 ng/L/s) in linear mode (B), which covers low, medium and high PRA patient samples.

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average PRA values obtained for the low, medium and high PRA pools in the intraday precision study were 0.08, 0.36, and 5.67 ng/L/s, respectively, when analyzed in the reflector mode, and 0.09, 0.42, and 6.07 ng/L/s, respectively, when analyzed in the linear mode. Intraday %CVs for low, medium and high PRA pools were 2.6%, 2.0% and 6.1%, respectively, when analyzed in the reflector mode, and 7.4%, 2.5%, and 1.5%, respectively, when analyzed in the linear mode (Fig. 5A). The average PRA values obtained for the low, medium, and high PRA pools in the interday precision study were 0.07, 0.35 and 5.24 ng/L/s, respectively, in the reflector mode, and 0.08, 0.40, and 5.06 ng/L/s, respectively, in the linear mode. Compared to the intraday precision study, the %CVs obtained in the interday precision study were higher in all cases except for the high PRA pool analyzed in the reflector mode. The %CVs of the interday precision study were 9.7%, 6.0%, and 2.6%, respectively, for the reflector mode, and 14.3%, 6.7%, and 12.6%, respectively, in the linear mode for low, medium, and high PRA pools (Fig. 5B). Both the reflector and linear mode iMALDI analyses result in comparable PRA values which fall within the predefined PRA ranges [21] for low, medium, and high PRA samples. However, the reflector mode tends to result in a significantly better precision for the low PRA pool and slightly improved precision for the medium PRA pool in both the intra- and interday precision study (Fig. 5A and B). The precision of the high PRA samples tends to be better in the linear mode for the intraday precision study, but better in the reflector mode in the interday precision study. Since the interday precision study is a better reflection of the routine day-to-day analysis, these results should be regarded as being more significant. The lower precision in the linear mode agrees with the findings by Anderson et al. [37]. Because patients suffering from primary aldosteronism show low PRA values due to high aldosterone concentrations (and therefore inhibited renin secretion), the clinical importance of this assay lies in accurately and precisely determining low and medium PRA values, whereas high PRA values are only of minor importance for diagnosing primary aldosteronism. In summary, the reflector mode leads to a higher precision than the linear mode. However, all of the %CVs obtained with either measurement mode fall within the precision recommendations of b 15% stated in the FDA guidelines for bioanalytical method validation [39], indicating that both measurement modes are precise enough for clinical utility.

(0.230 ng/L/s) (Fig. 6A). However, the two values differ by only ~10%. The precision of the automated and manual sample preparation is very good and comparable, with 3.0% CV with automated sample preparation, and 2.8% CV with manual sample preparation. In the linear mode, the average PRA value obtained with manual sample preparation (0.243 ng/L/s) was significantly lower (p = 0.005) than the average PRA value with automated sample preparation (0.263 ng/L/s) (Fig. 6B), but the magnitude of the relative difference was b 10%. The precision of the automated and manual sample preparation methods, as measured in the linear mode, are comparable (5.8% and 8.7%), but slightly higher than when the reflector mode was used. This lower precision can be explained by the lower resolution of the linear mode [37]. Compared to the manual sample preparation, automating the liquid handling steps significantly reduced the sample preparation time, which increases sample throughput and reduces the overall assay time. Additionally, automating the sample preparation reduces potential errors introduced by the personnel carrying out the assay, and also reduces the workload. For the 13 replicate plasma samples and one calibration curve (96 captures) prepared, the automation of the sample preparation reduced the duration of the “plasma preparation” step from ~ 11 min to 3.9 min, the “bead and standard transfer” step from ~27 min to 11.8 min, the “bead washing and spotting” step from ~ 46 min to 14.6 min, and the “matrix spotting” step from ~ 22 min to 2.5 min. For the full procedure (Fig. 7), which is designed for 29 patient samples and a calibration curve (192 captures), the Bravo protocols for the steps “plasma preparation”, “bead washing and spotting”, and “matrix spotting” can be performed within the same time as for the 13 replicates and one calibration curve, while the manual procedure would require twice the time for each of the four sample preparation steps. For example, the “bead washing and spotting” step would take approximately 1.5 h for 29 patient samples when performed manually, but only 14.6 min when performed on the Agilent Bravo. The overall time for performing the automated sample preparation steps manually for 29 patient samples and a calibration curve (192 captures) is ~ 3.5 h, but only ~ 0.5 h when performed in an automated fashion on the Agilent Bravo.

3.3. Comparison of manual and automated sample preparation

3.4.1. PRA determination for 188 patient samples Passing–Bablok regression of PRA values determined by iMALDI in the reflector and linear mode versus LC–MS/MS reference values showed a strong correlation with coefficients of determination (R2) of 0.94 in the reflector mode and 0.98 in the linear mode (Fig. 8A and B), with slopes of 0.59 and 0.67, respectively. Because the same plasmato-Ang I generation buffer volume ratios, and the same pH and composition of the Ang I generation buffer were used for the iMALDI and the LC–MS/MS sample preparation, we expected the slope to be 1.0.

A comparison of the fully manual sample preparation procedure for PRA determination by iMALDI with the automated sample preparation was performed on 13 aliquots of healthy control human plasma. MALDI analysis was performed in the reflector and linear mode. In the reflector mode, the average PRA value obtained by manual sample preparation (0.256 ng/L/s) was significantly higher (p = 0.0001) than the average PRA value obtained by automated sample preparation

3.4. Method comparison: PRA determination by iMALDI vs. LC–MS/MS

Fig. 5. Intraday precision (A) and interday precision (B) testing on low, medium and high PRA plasma pools.

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A) Reflector mode 0.32

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Preparation method Fig. 6. Comparison of automated and manual sample preparation for 13 replicates of generic human plasma by iMALDI in reflector mode (A) and linear mode (B).

When the Ang I concentrations of the 4 °C plasma aliquots determined by LC–MS/MS or by iMALDI are plotted against each other, or when the Ang I concentrations of the 37 °C plasma aliquots determined by these two methods are plotted against each other, the same slope of 0.6 is produced. This rules out the Ang I generation step — and thus the incubation step or the generation buffer — as the source of this difference (Supplementary Information Fig. S6). To eliminate the Ang I Nat and SIS stock solutions that were used for the iMALDI and LC–MS/MS methods as the cause of this difference, the stock solutions used for the iMALDI procedure were replaced with the stock solutions used for the LC–MS/MS procedure on the LC–MS/MS platform. This resulted in comparable PRA values (data not shown) and showed that the stock solutions were not responsible for the difference in the measurements. It might be possible, however, that the way the calibrators and internal standard were prepared in the two methods caused this systematic difference in the amounts of Ang I quantified. In the LC–MS/MS method, the Ang I SIS is pre-diluted in H2O and further diluted in 10% formic acid, with subsequent solid phase extraction. In the iMALDI method, the Ang I SIS is diluted in PBS (with 0.015% CHAPS) and 12 μL of bead–antibody conjugates (diluted in PBS with 0.015% CHAPS) are added prior to the affinity-capture step. In both cases, the SIS peptide is added to the sample after the Ang I generation period so its solvent does not affect the pH during the incubation step. Because the SIS peptides are added prior to the purification steps, which occur at different pHs, both the Nat and SIS forms should be equally affected. These two separation methods are currently incompatible — formic acid would interfere with the affinity capture. However, both the Nat and the SIS forms should be equally

Fig. 7. Comparison of durations of manual versus automated liquid handling steps of the sample preparation for 29 patient samples and one calibration curve (192 captures).

affected during the purification process. There could also be some possible interference in actual MS analysis, but this has not been proven. Whatever the source of the difference, because of the good correlation between the values produced by the iMALDI method and the LC–MS/MS reference method (which is already in clinical use), the difference in the measured amounts of Ang I can easily be adjusted by applying a correction factor determined from the slope of the method-comparison curves. The accuracy of this approach could be monitored by running quality control samples with every batch of samples. Of the PRA values determined in the reflector mode, 115/188 (61.2%) fall within the linear range of the assay (0.08–5.3 ng/L/s), whereas 3/188 PRA values (1.6%) are above, and 70/188 PRA values (37.2%) are below the linear range, respectively (Fig. 9). Of the 188 PRA values, 106 (56.3%), 70 (37.2%) and 12 (6.4%) fall within the low, medium, and high PRA categories, respectively. In comparison, 151/ 188 PRA values (80.3%) determined in the linear mode fall within the linear range of the linear mode iMALDI PRA assay (0.04–5.3 ng/L/s); 34/188 (18.1%) PRA values are below, and 3/188 (1.6%) values are above the linear range (Fig. 9). The increased number of linear mode PRA values falling within the linear range compared to the reflector mode data can be explained by the extended linear range at the low end of the linear mode iMALDI assay. Thus, using the linear mode instead of the reflector mode allows PRA determination for a larger number of low PRA patient samples. However, the sensitivity of both the reflector and linear mode is adequate for clinical utility. The distribution of the PRA values determined in the linear mode into low, medium, and high PRA categories with 102/188 (54.3%), 71/ 188 (37.8%) and 15/188 (8.0%), respectively, is comparable to the reflector mode results (Fig. 9), indicating comparable PRA outcomes for the reflector and linear mode data. In both the reflector and linear mode, only 3/188 (1.6%) values are above the linear range. The PRA values determined for these three samples are closer to the regression line when determined in linear mode (highest three values, Fig. 8B) as compared to the reflector mode (highest three values, Fig. 8A). This might indicate that for patient samples outside the upper end of the linear range the linear mode performs better than the reflector mode. However, because patients suffering from primary aldosteronism show low to medium PRA values, the high PRA range is of low importance for diagnosing primary aldosteronism. Furthermore, even though 70/188 PRA values determined in the reflector mode, and 34/188 PRA values determined in the linear mode are below the corresponding linear ranges (Fig. 9), these values are clinically useful. Because the LLOQs determined for the reflector (0.08 ng/L/s) and linear (0.04 ng/L/s) mode iMALDI PRA assay are in agreement with the Endocrine Society's Clinical Practice Guidelines which suggest

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Fig. 8. Method comparison: PRA determination of 188 samples by iMALDI and LC–MS/MS; Passing–Bablok regression plots for all 188 samples measured by MALDI in the reflector mode (A) and linear mode (B); Passing–Bablok regression plots for samples falling within the linear range of the reflector mode (C) and linear mode (D) iMALDI PRA assays, and their corresponding difference plots (E and F); gray bands = 95% confidence intervals.

a minimum sensitivity of 0.6–0.08 ng/L/s for PRA assays [14], the values determined to be below the linear range can be stated as “bLLOQ” and the actual LLOQ value is used for calculating the aldosterone–renin ratio (ARR). To determine the assay performance within the linear range of the iMALDI assay, the PRA values within the linear range (115/188 for the reflector mode; 151/188 for the linear mode) were plotted against the

corresponding LC–MS/MS values. Looking only at values within the linear range of the iMALDI PRA assay instead of using all 188 patient samples (note that all corresponding LC–MS/MS values fell within the linear range of the LC–MS/MS method: 0.03–9.26 ng/L/s) improved the R2 value of the reflector mode data from 0.94 (Fig. 8A) to 0.98 (Fig. 8C), and the R2 value of the linear mode from 0.98 (Fig. 8B) to 0.99 (Fig. 8D), indicating excellent correlation with the clinically

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Fig. 9. Distribution of PRA values determined by iMALDI in the reflector and linear mode into “above, within, or below the corresponding linear range”, and into “low, medium, and high PRA ranges”.

established LC–MS/MS PRA assay within the linear range of the iMALDI assay. Additionally, the difference plots comparing the iMALDI PRA values obtained in the reflector mode (Fig. 8E) and linear mode (Fig. 8F) to the LC–MS/MS reference values support the strong correlation between the iMALDI and LC–MS/MS methods. For the reflector and linear mode data, 114 of the 115 samples (99.1%; Fig. 8E), and 149 of the 151 samples (98.7%; Fig. 8F) fall within the 95% confidence interval. The mean %-difference of approximately − 50% reflects the trend observed in the corresponding Passing–Bablok regression curves (Fig. 8C and D), which indicate that the PRA values determined by iMALDI are approximately 60% of the LC–MS/MS values. However, even though the iMALDI-determined PRA values are lower, the correlation is excellent which suggests that a correction factor could be applied. 3.4.2. Throughput comparison Assuming optimized sample preparation using robotic systems and time-delayed preparation of multiple sample batches, which is possible for both the iMALDI and LC–MS/MS PRA assay, the limiting factor is the analysis times on the MALDI and LC–MS/MS systems. The analysis time of the LC–MS/MS method is 6 min per injection [22]. For each patient sample, one plasma aliquot is prepared at 4 °C and one is prepared at 37 °C, which results in an overall analysis time of 12 min per patient sample. In comparison, the analysis time of each MALDI spot takes 16 s. The current automated setup of the iMALDI procedure prepares the 4 °C and 37 °C plasma aliquots of each patient sample in triplicate, resulting in an analysis time of 1.6 min per patient. For 29 patient samples, the LC–MS/MS approach requires 348 min of analysis time, whereas the MALDI analysis only requires 46.4 min, which, with the current parameters of the iMALDI PRA assay, makes iMALDI analysis 7.5 times as fast as the LC–MS/MS analysis. This demonstrates the potential of iMALDI as a high-throughput method for largescale routine clinical analysis or biomarker validation studies as this shorter analysis time becomes especially important for larger numbers of samples. 3.5. Potential for significant throughput increases of the iMALDI PRA assay To make the iMALDI PRA assay into a large-scale assay, the current setup of the assay, which allows PRA determination of 29 patient samples (192 captures) per day, needs to be further improved. First of all, only 192 of the 384 spots of the four MSP MALDI targets are currently being used (Fig. 2). Therefore, 384 spots, which correspond to 61 patient samples and a calibration curve with the current iMALDI PRA assay setup, could theoretically be run in a single MALDI analysis. Second, the measurement of the 4 °C plasma aliquot (blank) appears to be unnecessary for accurate PRA determination when the reflector mode is used for MALDI analysis. Instead, only the Ang I concentration determined for the 37 °C plasma aliquot is required. The validity of

this approach has been shown in recent LC–MS/MS studies [24,40]. This approach seems to be valid if appropriate pre-analytical conditions are used, which keep the Ang I concentrations at a minimum before the 37 °C incubation [41], and when suitable Ang I generation times are used (up to 18 hour incubation times have been used for low plasma renin samples [42]). Furthermore, whereas RIAs require the blank measurement in order to account for cross-reactive species interfering with detection of the target analyte, mass spectrometric methods such as iMALDI allow differentiation of the analyte of interest from crossreacting species by their mass differences. Additionally, Ang I concentrations in the blank are often below the LLOQ [24]. This was also observed in our own method comparison for PRA determination by iMALDI and LC–MS/MS for 188 patient samples, where only 3 of the 188 patient samples measured in the reflector mode, and 9 of the 188 patient samples measured in the linear mode showed Ang I concentrations in the 4 °C plasma aliquots above the corresponding LLOQs (data not shown). To test the validity of eliminating the 4 °C plasma aliquot and only using the 37 °C plasma Ang I concentration for PRA calculation in the iMALDI PRA assay, the method comparison data collected for the 188 patient samples by iMALDI in both the linear and reflector modes was used. For the patient samples falling within the linear range of the iMALDI PRA assay (115 of the 188 in the reflector mode, and 151 of the 188 in the linear mode), the PRA values were calculated by either factoring in both the 4 °C (blank) and 37 °C plasma Ang I concentrations, or by only using the 37 °C plasma Ang I concentration (Fig. 10). For both the reflector mode and linear modes, both calculation methods showed excellent correlation with R2 values of 0.9999 and 0.9988, and slopes of 1.02, respectively (Fig. 10A and B). The difference plot for the reflector mode data shows that all PRA values fall within ± 5%, and that 107 of the 115 PRA values (93.0%) fall within the 95% confidence interval (Fig. 10C). The difference plot for the linear mode data shows that 148/151 PRA values (98.0%) fall within ±25%, whereas 3/151 PRA values (2.0%) show up to a −120% difference. Additionally, 147 of the 151 (97.4%) PRA values fall within the 95% confidence interval. The wider spread of the linear mode PRA values can be explained by the lower precision of the linear mode. Both difference plots show that with smaller PRA values, the %-difference between both calculation methods increases. This can be explained by the increased influence of the Ang I concentration of the 4 °C plasma sample on the calculated PRA value for low renin concentrations, because the blank cannot be measured accurately. In conclusion, the data suggests that when the reflector mode is used for PRA determination, the blank measurement for PRA is insignificant and can be eliminated. This step alone would increase the sample throughput from 29 to 58 patient samples (192 captures) analyzed in the same amount of time. Third, the current setup of the iMALDI PRA assay requires triplicate captures for each calibration curve point and for each 4 °C and 37 °C plasma sample. To assess the possibility of reducing the triplicate captures to single captures, the data collected for the 188 sample method comparison was analyzed in more detail. The %CVs calculated for the Nat/SIS ratios of the triplicate 37 °C plasma sample captures falling within the linear range of the iMALDI PRA assay were calculated and plotted in Fig. 11. In the reflector mode, the Nat/SIS ratios for 114 of the 115 of the 37 °C plasma samples (99.1%) showed b10% CV, and 1 of the 115 samples (0.9%) showed a %CV between 10 and 15%. In the linear mode, 100/151 samples (59.6%) showed b 10% CV, 24 of the 151 samples (15.9%) showed 10–15% CV, and 37 of the 151 samples (24.5%) showed CVs of N15%, with up to 127% for one 37 °C plasma sample. These data suggest that the triplicate captures can be reduced to single captures when the reflector mode is used for the MALDI analysis. Compared to the current setup (using 4 °C and 37 °C plasma captures), this would increase the sample throughput from 29 to 93 patient samples (192 captures) in 46.4 min. Furthermore, with the current setup, only ~30 min of robot time are needed for preparing 29 patient samples (192 captures) for MALDI

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Fig. 10. Correlation of PRA values determined by iMALDI, either calculated with the blank (4 °C plasma) Ang I concentration, or with the blank Ang I concentration set to 0 fmol.

analysis. This means that only 2.1% of total robot time capacity per day is being used to run the assay. This suggests that multiple batches of samples can be run slightly off-set from each other to utilize the currently unused robot time. With approximately 6 h of total assay time prior to MALDI analysis, a realistic schedule would be to run 4 batches off-set by approximately 30 min, using 1 calibration curve. With the current setup, this would increase the sample throughput from 29 samples and one calibration curve to 125 samples. Since MALDI preparations are known to be stable for weeks when protected from light [36], the sample preparation does not require direct subsequent MALDI analysis.

The use of an internal standard (Ang I SIS) also reduces the effect of potential degradation on Ang I quantitation in a patient samples. Taken together, the elimination of the 4 °C plasma aliquot (blank) measurement and reducing the triplicate analyses to a single analysis would increase the number of samples prepared from 29 to 186 samples (192 captures) within ~6 h, followed by approximately 1 h of analysis. Furthermore, performing the “off-set” sample preparation for four batches of samples per day would increase the number of samples analyzed to 744 samples per day, which would be an approximately 25-fold increase in throughput. In summary, use of the reflector mode for MALDI analysis would allow a significant increase in the sample throughput. 4. Conclusions

Fig. 11. %CVs calculated for the Nat/SIS ratios of the triplicate 37 °C plasma sample captures of the 188 iMALDI versus LC–MS/MS method comparison samples falling within the linear range of the iMALDI PRA assay.

We have developed a high-throughput iMALDI PRA assay with automated liquid handling which enables the preparation and analysis of 192 captures (29 patient samples and one calibration curve) within ~6–7 h. It shows excellent correlation with a clinically established LC– MS/MS method for PRA determination at significantly faster analysis times (~ 7.5-fold). A comparison of the reflector and linear mode MALDI analysis indicates that the clinical iMALDI PRA assay should ideally be run in the reflector mode. It covers the clinically relevant range of PRA patient samples, and shows increased precision (2.0–9.7%) compared to the linear mode. It also allows elimination of the 4 °C plasma (blank) incubation and the reduction of triplicate to single captures per patient sample, thereby significantly increasing sample throughput for the PRA assay up to a maximum of 744 samples per day. Assay validation was performed with a single lot of antibody. However, a second

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lot was tested and found to show comparable sensitivity and a significantly lower contaminant signal at m/z 1295.7 (data not shown). In order to circumvent the possibility of batch-to-batch variability — or the possibility of running out of the polyclonal antibody — the next step would be to investigate the performance of anti-Ang I monoclonal antibodies. An important result of this study is that an iMALDI platform with automated liquid handling has now been established and can be used to perform iMALDI assays for additional clinical analytes.

[16] [17]

[18] [19]

Conflict of interest [20]

CHB is the sole inventor on the patent for the iMALDI technology. The other authors declare no conflict of interest. Acknowledgements The authors would like to thank Genome Canada and Genome British Columbia for funding and support through the “Science and Technology Innovation Centre (S&TIC).” Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.10.008.

[21]

[22]

[23]

[24]

[25] [26]

[27]

References [1] World Health Organization, Global Status Report on Noncommunicable Diseases 2010, A. Alwan, Geneva, 2011. 1–162. [2] N. Sukor, Primary aldosteronism: from bench to bedside, Endocrine 41 (2012) 31–39. [3] G.P. Rossi, L.A. Sechi, G. Giacchetti, V. Ronconi, P. Strazzuo, J.W. Funder, Primary aldosteronism: cardiovascular, renal and metabolic implications, Trends Endocrinol. Metab. 19 (2008) 88–90. [4] P. Milliez, X. Girerd, P.F. Plouin, J. Blacher, M.E. Safar, J.J. Mourad, Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism, J. Am. Coll. Cardiol. 45 (2005) 1243–1248. [5] C. Catena, G. Colussi, E. Nadalini, A. Chiuch, S. Baroselli, R. Lapenna, L.A. Sechi, Cardiovascular outcomes in patients with primary aldosteronism after treatment, Arch. Intern. Med. 168 (2008) 80–85. [6] R.M. Carey, Primary aldosteronism, J. Surg. Oncol. 106 (2012) 575–579. [7] D.J. Campbell, J. Nussberger, M. Stowasser, A.H.J. Danser, A. Morganti, E. Frandsen, J. Menard, Activity assays and immunoassays for plasma renin and prorenin: information provided and precautions necessary for accurate measurement, Clin. Chem. 55 (2009) 867–877. [8] S.A. Atlas, The renin–angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition, J. Manag. Care Pharm. 13 (2007) 9–20. [9] P. Gupta, R. Francosaenz, P.J. Mulrow, Locally generated angiotensin-II in the adrenal-gland regulates nasal, corticotropin-stimulated, and potassium-stimulated aldosterone secretion, Hypertension 25 (1995) 443–448. [10] H.P. Rang, M.M. Dale, J.M. Ritter, R.J. Flower, G. Henderson, Rang & Dale's Pharmacology, 7th edition Elsevier Health Sciences, UK, 2011. [11] J.W. Conn, D.R. Rovner, E.L. Cohen, Suppression of plasma renin activity in primary aldosteronism — distinguishing primary from secondary aldosteronism in hypertensive disease, JAMA 190 (1964) 213–221. [12] C. Volpe, A. Hoog, T. Ogishima, K. Mukai, M. Lu, M. Thoren, B. Hamberger, Immunohistochemistry improves histopathologic diagnosis in primary aldosteronism, J. Clin. Pathol. 66 (2013). [13] W.F. Young, Primary aldosteronism: renaissance of a syndrome, Clin. Endocrinol. 66 (2007) 607–618. [14] J.W. Funder, R.M. Carey, C. Fardella, C.E. Gomez-Sanchez, F. Mantero, M. Stowasser, W.F. Young, V.M. Montori, Case detection, diagnosis, and treatment of patients with primary aldosteronism: an endocrine society clinical practice guideline, J. Clin. Endocrinol. Metab. 93 (2008) 3266–3281. [15] K. Hiramatsu, T. Yamada, Y. Yukimura, I. Komiya, K. Ichikawa, M. Ishihara, H. Nagata, T. Izumiyama, A screening test to identify aldosterone producing adenoma by measuring

[28]

[29] [30]

[31]

[32]

[33] [34] [35]

[36]

[37]

[38] [39] [40]

[41] [42]

plasma renin activity results in hypertensive patients, Arch. Intern. Med. 141 (1981) 1589–1593. M. Stowasser, A.H. Ahmed, E. Pimenta, P.J. Taylor, R.D. Gordon, Factors affecting the aldosterone/renin ratio, Horm. Metab. Res. 44 (2012) 170–176. H.S. Willenberg, O. Vonend, M. Schott, X. Gao, D. Blondin, A. Saleh, L.C. Rump, W.A. Scherbaum, Comparison of the saline infusion test and the fludrocortisone suppression test for the diagnosis of primary aldosteronism, Horm. Metab. Res. 44 (2012) 527–532. G.W. Boyd, A.E. Fitz, A.R. Adamson, W.S. Peart, Radioimmunoassay determination of plasma-renin activity, Lancet 1 (1969) 213–218. S. Oparil, T.J. Koerner, E. Haber, Effects of pH and enzyme inhibitors on apparent generation of angiotensin I in human plasma, J. Clin. Endocrinol. Metab. 39 (1974) 965–968. F. Fyhrquist, L. Puutula, Faster radioimmunoassay of angiotensin I at 37 °C, Clin. Chem. 24 (1978) 115–118. M.H. Alderman, H.W. Cohen, J.E. Sealey, J.H. Laragh, Plasma renin activity levels in hypertensive persons: their wide range and lack of suppression in diabetic and in most elderly patients, Am. J. Hypertens. 17 (2004) 1–7. A.G. Camenzind, J.G. van der Gugten, R. Popp, D.T. Holmes, C.H. Borchers, Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension, Clin. Proteomics 10 (2013) 20. D.L. Chappell, T. McAvoy, B. Weiss, R. Weiner, O.F. Laterza, Development and validation of an ultra-sensitive method for the measurement of plasma renin activity in human plasma via LC–MS/MS, Bioanalysis 4 (2012) 2843–2850. S. Carter, L.J. Owen, M.N. Kerstens, R.P.F. Dullaart, B.G. Keevil, A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solidphase extraction, Ann. Clin. Biochem. 49 (2012) 570–579. S.K. Grebe, R.J. Singh, LC–MS/MS in the clinical laboratory — where to from here? Clin. Biochem. Rev. 32 (2011) 5–31. J. Jiang, C.E. Parker, K.A. Hoadley, C.M. Perou, G. Boysen, C.H. Borchers, Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosis, Proteomics Clin. Appl. 1 (2007) 1651–1659. J. Jiang, C.E. Parker, J.R. Fuller, T.H. Kawula, C.H. Borchers, An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis, Anal. Chim. Acta. 605 (2007) 70–79. E.N. Warren, J. Jiang, C.E. Parker, C.H. Borchers, A method for the absolute quantitation of cancer-related proteins using a MS-based peptide chip, BioTechniques 38 (2005) S7–S11. C.H. Borchers, C.E. Parker, Improving the biomarker pipeline, Clin. Chem. 56 (2010) 1786–1788. C.H. Borchers, Methods of Quantitation and Identification of Peptides and Proteins, in: Canadian_Patent_Office (Ed.), Canadian Patent Office, The University of North Carolina at Chapel Hill, Canada, 2011. A. Cherkaoui, J. Hibbs, S. Emonet, M. Tangomo, M. Girard, P. Francois, J. Schrenzel, Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level, J. Clin. Microbiol. 48 (2010) 1169–1175. O. Garner, A. Mochon, J. Branda, C.A. Burnham, M. Bythrow, M. Ferraro, C. Ginocchio, R. Jennemann, R. Manji, G.W. Procop, S. Richter, J. Rychert, L. Sercia, L. Westblade, M. Lewinski, Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system, Clin. Microbiol. Infect. 20 (2014) 335–339. S. Biswas, J.M. Rolain, Use of MALDI-TOF mass spectrometry for identification of bacteria that are difficult to culture, J. Microbiol. Methods 92 (2013) 14–24. Bruker Daltonics, MALDI Biotyper CA System, 2013. M.A. Kuzyk, D. Smith, J.C. Yang, T.J. Cross, A.M. Jackson, D.B. Hardie, N.L. Anderson, C.H. Borchers, Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma, Mol. Cell. Proteomics 8 (2009) 1860–1877. K. Benkali, P. Marquet, J. Rerolle, Y. Le Meur, L. Gastinel, A new strategy for faster urinary biomarkers identification by nano-LC–MALDI-TOF/TOF mass spectrometry, BMC Genomics 9 (2008) 541. N.L. Anderson, M. Razavi, T.W. Pearson, G. Kruppa, R. Paape, D. Suckau, Precision of heavy-light peptide ratios measured by MALDI-TOF mass spectrometry, J. Proteome Res. 11 (2012) 1868–1878. J.E. Sealey, R.D. Gordon, F. Mantero, Plasma renin and aldosterone measurements in low renin hypertensive states, Trends Endocrinol. Metab. 16 (2005) 86–91. Food and Drug Administration, Guidance for Industry — Bioanalytical Method Validation, 2001. C.E. Bystrom, W. Salameh, R. Reitz, N.J. Clarke, Plasma renin activity by LC–MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity, Clin. Chem. 56 (2010) 1561–1569. J. Brossaud, J.-B. Corcuff, Pre-analytical and analytical considerations for the determination of plasma renin activity, Clin. Chim. Acta 410 (2009) 90–92. J.E. Sealey, Plasma renin activity and plasma prorenin assays, Clin. Chem. 37 (1991) 1811–1819.