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Duplexed iMALDI for the detection of angiotensin I and angiotensin II
Methods 56 (2012) 213–222
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Duplexed iMALDI for the detection of angiotensin I and angiotensin II D. Randal Mason a, Jennifer D. Reid a, Alexander G. Camenzind a, Daniel T. Holmes b, Christoph H. Borchers a,c,⇑ a
University of Victoria-Genome British Columbia Proteomics Centre, Victoria, British Columbia, Canada V8Z 7X8 University of British Columbia, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, 1081 Burrard St., Vancouver, British Columbia, Canada V6Z 1Y6 c University of Victoria, Department of Biochemistry and Microbiology, Victoria, British Columbia, Canada V8Z 7X8 b
a r t i c l e
i n f o
Article history: Available online 25 February 2012 Keywords: Angiotensin I Angiotensin II Plasma renin activity ACE activity iMALDI Hypertension Immunoaffinity mass spectrometry Duplex iMALDI
a b s t r a c t An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a 50 lL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of 10 amol/lL (or 13 pg/mL AngI and 11 pg/ mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-lL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4–100 fmol/lL (5–130 ng/mL) and 100–2500 amol/lL (106–2614 pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction 1.1. Renin-angiotensin system Discovered over 100 years ago, the renin-angiotensin system (RAS) is a biochemical pathway which plays a critical role in cardiovascular physiology (Scheme 1) [1]. The pathway begins with angiotensinogen, an a-2 globulin constitutively manufactured by
the liver [1]. Of the 452 amino acids in its sequence, only the first 10 are known to play a role in RAS, and angiotensinogen itself has no known independent function [2]. Renin is the next critical RAS protein. Its enzymatic precursor, prorenin, is secreted into the bloodstream from the juxtaglomerular apparatus of kidneys. Once it has been activated to renin, it cleaves the decapeptide angiotensin I (AngI, DRVYIHPFHL) [3] from angiotensinogen. Like angiotensinogen, AngI itself has no known
Scheme 1. RAS overview.
Abbreviations: ACE, angiotensin-converting enzyme; AngI, angiotensin I; AngII, angiotensin II; iMALDI, immunoMALDI (immuno-Matrix-Assisted Laser Desorption/ Ionization); PRA, plasma renin activity; RAS, renin-angiotensin system; SIS, stable-isotope-labeled internal standard; LOD, Limit of Detection; S/N, signal-to-noise ratio; MS, mass spectrometry; MS/MS, tandem mass spectrometry; CV, coefficient of variation; PAR, peak area ratio. ⇑ Corresponding author. Address: University of Victoria-Genome British Columbia Proteomics Centre, 3101-4464 Markham St., Victoria, British Columbia, Canada V8Z 7X8. Fax: +1 250 483 3238. E-mail address: [email protected] (C.H. Borchers), [email protected] (C.H. Borchers). 1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2012.02.006
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Add Labeled Angiotensin I and II Peptide Standards
Whole Plasma
Angiotensin II Angiotensin I Add Antiangiotensin Affinity Beads
MS
m/z
MS2
analyze
Angiotensin Capture Period m/z
Fig. 1. iMALDI workflow schematic.
physiological activity but is the substrate for angiotensin converting enzyme (ACE). ACE is a metalloprotease, which cleaves two Cterminal residues from AngI to produce angiotensin II (AngII, DRVYIHPF), the primary physiological effector of the RAS pathway, acting as an endocrine, paracrine/autocrine and intracrine hormone [4]. In recent years, a number of different Ang receptors, expressed by different tissues, have been identified for which AngII is a ligand [5]. Binding of the AngII ligand to these receptors increases blood pressure directly by vasoconstriction and indirectly by stimulating the manufacture and release of aldosterone, anti-diuretic hormone (ADH) and through stimulation of the sympathetic nervous system [6].
1.2. RAS and disease states Physiological derangements of the RAS may arise from within the system itself, for example, through acquired or inherited mutation leading to alteration in the function and/or abundance of its various components. An imbalance in RAS may also be a response to changes in related physiological systems such as mechanical stress to the body, environmental or dietary changes or some other primary disease [4]. Traditionally, pathological upregulation of RAS (e.g., from renal arterial stenosis, primary renal parenchymal diseases, estrogen effect) has been associated primarily with cardiovascular risk due to the hypertensive effects of AngII [7]. More recently, the RAS has been implicated in a wide range of conditions from musculoskeletal diseases, to Alzheimer’s to various cancers. In cancers, changes in RAS activity are associated with tumor angiogenesis, which is required for continued tumor growth. RAS enzyme inhibitors have been shown to have anti-tumor effects [8]. With respect to Alzheimer’s disease, there is an epidemiological correlation between hypertension and the development of this disease [9]. It has also been recently discovered that the brain itself has a local RAS that may mediate the development of Alzheimer’s disease [6,9]. Cardiovascular diseases are the number one killer globally, with an annual combined (direct and indirect) treatment cost that exceeds $430 billion dollars
[10,11]. Therefore, because of its association with all of these diseases, the biochemical investigation and therapeutic regulation of RAS is of crucial importance to public health. 1.3. Methods of assessing RAS The RAS is routinely monitored by measuring the concentrations of AngI or AngII, renin or ACE using competitive radioimmunoassay and ELISA. Mass spectrometry methods have also been developed for AngI and AngII, including an LC-MRM method [12]. Immunoradiometric and chemiluminometric assays are available for measuring the concentration of renin in plasma. Spectrophotometric assays are also used for determining ACE activity, where a synthetic substrate is used for the measurement. Functional enzymatic assays are also available, which determine the ability of the RAS system to regulate arterial pressure by measuring the enzymatic activity of renin or ACE. The low levels of Ang peptides in plasma has favored the utilization of functional enzymatic assays, whereby a standardized incubation period is used to increase the levels of the Ang peptides above those that would occur endogenously in order to enable their detection. The plasma renin activity is the most common method of assessing RAS function and measures the production of AngI as a product of angiotensinogen cleavage by renin. As biochemical methods have evolved, the assessment of renin activity moved from bioassay methods to the radioimmunoassay methods that are still commonly in use today [13,14]. 1.4. The iMALDI method iMALDI is a technique that involves affinity capture of the peptide of interest on anti-peptide Ab-coated beads, which are washed to remove non-specifically-bound interferences (Fig. 1) [15–19]. After washing, the beads are spotted directly onto a MALDI target plate for mass spectrometric analysis. The beads remain on the target. There is no prior elution of the analyte – the analyte is eluted by the matrix solution. The principle of immunocapture is also used in SISCAPA (Stable Isotope Standards and Capture by Anti-
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Peptide Antibodies, [20]) and in some versions of SELDI (SurfaceEnhanced Laser Desorption/Ionization [21]). SISCAPA and iMALDI both use anti-peptide antibodies for affinity purification and cocapture of stable-isotope-labeled standards for quantitation. However, in iMALDI, the analytes are not eluted from the beads and MALDI is used for the analysis. The SELDI approach relies on antibodies (not necessarily anti-peptide antibodies) but in SELDI, the surface of the MALDI target is modified with these capture agents. iMALDI has the advantage of using standard MALDI targets, and varying amounts of affinity beads can be placed on the target used to produce the required detection sensitivity. Multiple iMALDI assays, with different target peptides, can be done on a single target, leading to a very flexible analytical platform, with MALDI-MS/MS providing the needed specificity. Another advantage of iMALDI is that it is a simple methodology that lends well to automation, producing assays that are inexpensive, sensitive, and specific. Due to the unique masses and sequences of the target peptides, mass spectrometric analysis provides attomole-level LODs on-target (using only 50 lL of plasma) and with very high, if not absolute, specificity of detection. In addition, once the bead-Ab-peptide conjugates are spotted on the target, the analyte can remain stable for months and be reanalyzed if necessary. The iMALDI technique has proven to be effective in detecting: low-abundance bacterial proteins in human plasma [17], the presence of upregulated receptors in tumorigenic cells [16], and the detection of AngI alone in human plasma [18]. iMALDI also allows for the simultaneous detection (multiplexing) of multiple peptides from a common complex background.
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Synthetic AngI and AngII were purchased from Sigma–Aldrich. Isotopically labeled AngI and AngII were synthesized with a stable-isotope-coded arginine residue (+10 Da) (DRVYIHPFHL and DRVYIHPF, respectively) at the University of Victoria Genome BC Proteomics Centre (Victoria, BC, Canada), according to previouslydescribed protocols [23]. 2.2. Antibody-bead conjugation Magnetic Protein G Dynabeads were precipitated in the manufacturer’s detergent-containing storage buffer using a DynaMag-2 magnet (Invitrogen). The binding capacity of the Protein G Dynabeads was determined according to the manufacturer’s specifications, and an excess of beads was used in these iMALDI experiments to ensure maximum binding of the antibody. The supernatant was removed, and the beads were washed three times in 50 lL aliquots of 1 phosphate buffered saline (PBS) by vortexing the beads in buffer and removing the supernatant in order to discard as much of the storage buffer as possible. A 5 lL-aliquot of beads was used for each reaction. Antibodies (2 lL anti-AngI pAb or 1 lL anti-AngII pAb) were immobilized on Protein G Dynabeads in 1 PBS, pH 7.4 for 1 h at 4 °C. Following the 1-h incubation, excess antibody was removed by washing the beads three times in 1 PBS (50 lL). Beads were resuspended in 1 PBS (10 lL buffer per reaction) and then aliquoted into the individual samples. For the duplexed experiments, the anti-AngI pAb-bead conjugates and anti-AngII pAb-bead conjugates were combined at this stage. 2.3. PRA standards
1.5. Advantages of a duplexed iMALDI assay With regard to the RAS, we hypothesized that it should be possible to detect the presence and levels of both AngI and AngII in whole human plasma. The molecular weights of AngI and AngII are well-suited to iMALDI, (MW’s of 1295.6 Da and 1045.5 Da, respectively), which allows these peptides to be detected from plasma without the need for tryptic digestion, which is required in most proteomics experiments, and is normally included in the standard iMALDI workflow. With a duplexed iMALDI assay, it is possible to simultaneously quantify both AngI and AngII, in a single experiment, in a single sample. Because this is a molecular weight-based assay with mass spectrometric detection, there is no crossreactivity between AngI and AngII. The ability to simultaneously measure the levels of these two peptides may also aid in the monitoring of these different pathologies. Thus with the duplexed iMALDI approach, it is possible to simultaneously quantify AngI, AngII and, with incubation steps, renin and ACE activities also. 2. Methods 2.1. Chemicals and reagents Polyclonal antiserum (pAb) to AngI was obtained by immunizing rabbits with angiotensin-I/albumin complexes in Freund’s adjuvant [22]. Anti-AngII pAb (2 mg/mL) was purchased from BACHEM (Torrance, CA). The polyclonal anti-AngI antibody and the human plasma samples were obtained from the department of Pathology and Laboratory Medicine, St. Paul’s Hospital (Vancouver, BC, Canada). The anti-AngI pAb and anti-AngII pAb were both affinity purified antisera and as such, the antisera itself contains bound AngI and AngII peptides, respectively. Antibody-bead conjugation was performed using magnetic Protein G Dynabeads (30 mg/mL) (Invitrogen).
The samples used for these experiments were Lyphochek™ Hypertension Markers Control Levels 1, 2 and 3 from BIO-RAD (lot number 35940, Irvine, CA)., which consist of lyophilized human plasma. The BIO-RAD samples consist of processed lyophilized human plasma with added constituents of human and animal origin. They are used as quality-control markers for hypertension assays such as the DiaSorin Gamma Coat assay for plasma renin activity (DiaSorin Canada, Mississauga, ON, P2621). The standards have known DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60 and 10.7–21.7 ng/mL/h for the Level 1, Level 2, and Level 3 standards, respectively. The normal range for plasma renin activity is 0.73–3.96 ng/mL/h [24] which would place the Levels 1, 2, and 3 standards from BIO-RAD at the normal, high-normal, and high range for plasma renin activity, respectively. 2.4. MALDI instrument parameters MALDI MS and MS/MS data were acquired on an AB Sciex 4800 MALDI mass spectrometer. The instrument is equipped with a solid-state ND:YAG laser, operated at 200 Hz. The source was operated in the positive ion reflectron mode. The MS/MS setting was 2KV (positive). An average of 100 shots/sub-spectrum was acquired in the MS mode, with a total of 1000 shots per spectrum. In the MS/MS mode, 25 shots were acquired per subspectrum, with a total of 40 sub-spectra acquired and a total of 1000 total shots per spectrum. Delayed extraction was on, and the MS/MS fragmentation mechanism was CID. Applied Biosystems 4000-Series Explorer software was used for instrument control; Applied Biosystems Data Explorer was used for the Data Analysis. Peak areas were used for quantitation. 2.5. Duplexed iMALDI Following antibody-bead conjugation, the PBS supernatant was removed using the magnet and the beads were resuspended in 50 lL of either PBS or whole human plasma. The stable-isotope
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Table 1 The concentrations of stable isotope peptides spiked into 50 lL of whole human plasma to determine the Limit of Detection of the duplexed iMALDI assay. Sample
[AngI] amol/lL
[AngI] ng/mL
[AngII] amol/lL
[AngII] ng/mL
1 2 3 4 5
500 100 50 25 10
0.653 0.131 0.065 0.033 0.013
500 100 50 25 10
0.528 0.106 0.053 0.026 0.011
versions of AngI and AngII were added at known amounts to the samples to act as a reference standards. The samples were then incubated at 37 °C (4 °C for the control sample), where endogenous AngI and AngII and the internal standards were affinity-captured from the plasma solution by the antibody-bead conjugates. Following incubation, the beads were separated using the DynaMag-2 magnet, the supernatant was removed and the beads were washed four times in 25 mM ammonium bicarbonate (ABC). The samples were then re-suspended in 10 lL 25 mM ABC for the duplexed
Fig. 2. iMALDI mass spectra of 25 amol/lL AngI and AngII captured on affinity beads (a) MALDI-MS/MS and MALDI-MS spectra of AngI SIS (b) MALDI-MS/MS and MALDI-MS spectra of AngII SIS.
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Fig. 3. Linearity of the duplexed iMALDI assay as demonstrated by the addition of varying levels of the SIS peptides to a human plasma sample. (a) Heavy:light peptide peak area ratios for different levels of AngI (b) Heavy:light peptide peak area ratios for different levels of AngII. Source data is shown in Supplementary Data Table I.
samples (5lL for non-duplexed samples). A 1-lL aliquot of each sample was then spotted in triplicate onto a polished steel, 384-well MALDI plate. Once dry, each sample was coated with 1 lL of CHCA matrix (10 mM a-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% trifluoroacetic acid). The samples were then analyzed using an Applied Biosystems 4800 MALDI TOF/TOF mass spectrometer. 2.6. Assay validation 2.6.1. Linearity and quantitation Determination of the linearity of the iMALDI assay and quantitation were performed by adding standard amounts of the synthetic natural forms of AngI (20 fmol/lL; 26 ng/mL) and AngII (0.5 fmol/lL; 0.53 ng/mL), while varying the amount of the stable-isotope versions of AngI (0–100 fmol/lL; 0–130 ng/mL) and AngII (0–2.5 fmol/lL; 0–2.6 ng/mL) added to PBS buffer and whole human plasma. Using the standard-addition method, this generated a regression line from which the endogenous levels of AngI and AngII could be assessed [25]. The peak area ratios (PARs) of the natural and stable isotope forms of the peptide were used to quantify the natural form of the peptide and to detect an increase in endogenous levels over time when a known amount of stable isotope was introduced to the sample. 2.6.2. Limit of Detection for duplexed iMALDI The Limit of Detection (LOD) was determined by spiking known concentrations of the stable-isotope versions of AngI (m/z 1306.6) and AngII (m/z 1056.5) into a pooled whole human plasma sample. We had previously established that the stable isotope and natural forms of the peptides demonstrated equal affinities for their respective antibodies (data not shown). The duplexed approach described above was employed, with stable isotope standards of AngI and AngII being spiked into different plasma samples at the concentrations shown in Table 1. The samples were incubated for 2 h at 4 °C, then washed and analyzed via MALDI-TOF/TOF mass spectrometry. The LOD for the duplexed iMALDI assay was defined as the lowest concentration at which the peak associated with the SIS form of the Ang peptide could consistently be detected at a S/N of 2:1. Tandem mass spectrometric sequencing was performed on the samples containing the lowest detectable peaks to confirm the identities of the given peptides.
2.6.3. Detection and time course studies of AngI and AngII Lyphochek™ Hypertension Controls (Levels 1, 2, and 3, corresponding to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60, and 10.7–21.7 ng/mL/h) were used to track changes in the concentrations of AngI and AngII over time. This experiment was carried out without the addition of any enzymatic inhibitors, using 350 lL of plasma and stable isotopes at concentrations of 20 fmol/lL (26 ng/mL) and 500 amol/lL (0.53 ng/mL) for AngI and AngII, respectively, and the equivalent of seven 10-lL aliquots of antibody-bead conjugates. Following the incubation of plasma with the stable-isotope standards of AngI and AngII and antibody-bead conjugates for 3 h at 4 °C (to allow peptide binding), the samples were incubated at 37 °C to promote AngI and AngII generation. Fifty-microliters aliquots were taken from the sample at the following time points: 0, 4, 7, 12, 24, 48, and 99 h, the beads were washed, re-suspended, and analyzed as described above. The technical variability of the mass spectrometric portion of the assay was assessed by spotting the same sample in triplicate on the MALDI target plate. Each sample spot was measured five times, and the intra-spot coefficient of variation (CVs) and standard deviation were determined. The average of the five acquisitions is then used as a representative value for that spot, and the CV was then taken across three replicate sample spots. The LOD results were taken from the 0-h time point of this plasma incubation experiment where, with a 2-h incubation at 4 °C, there would have been a very low level of enzymatic activity in the plasma. 3. Results and discussion We have previously developed an iMALDI assay for AngI in human plasma [16]. This iMALDI approach also provided an improved LOD of synthetic angiotensin I in buffer by 80-fold compared to AngI spotted directly on a MALDI target. The LOD for AngI in these experiments was determined to be 65 pg/mL, thus meeting the clinical requirements for measuring low renin activity. Moreover, because of its high sensitivity, only 50 lL of plasma is needed for the iMALDI assay which is lower than that required for many automated immunoassay systems for various human proteins. Because of the sensitivity of this assay and the specificity of iMALDI, we hypothesized that it should be possible to develop an assay for the quantitation of both AngI and AngII, simultaneously. After incubation, the assay would also be valuable in identifying any perturbations in renin and/or ACE activity that might assist the clinician identifying a specific pathology.
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Fig. 4. (a) iMALDI spectrum showing both AngI and AngII, endogenous and SIS forms. (b) Detection of AngI in whole human plasma using duplexed iMALDI. The spectra labeled ‘‘Level 1’’, ‘‘Level 2’’, and ‘‘Level 3’’ are from three BIORAD standards (which correspond to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60, and 10.7– 21.7 ng/mL/h, respectively), while spectrum labeled ‘‘buffer’’ is the result of incubating the antibody beads in phosphate buffered saline (PBS); (c) detection of angiotensin II (AngII) in whole human plasma using the duplexed iMALDI, labeled as above; (d) simultaneous detection and relative quantitation of angiotensins I and II in BIO-RAD hypertension plasma standards through the duplexed iMALDI. The samples were incubated in 50 lL of plasma for 2 h at 4 °C to allow antibody capture of the peptides. The error bars reflect the CV determined by measuring the same sample in triplicate on different spots on the MALDI plate (L1, Level 1; L2, Level 2; and L3, Level 3; corresponding to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60 and 10.7–21.7 ng/mL/h, respectively).
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Fig. 4 (continued)
3.1. Determination of the LOD The Limit of Detection (LOD) of any iMALDI assay, including this duplexed assay, is highly dependent on the affinities of the antibodies used for the target peptides. LODs for this assay were defined as the lowest analyte concentration which could reliably be distinguished from background and could be confirmed by tandem MS/MS with a S/N cutoff of 2:1. As with other affinity-based techniques, such as SISCAPA, the LOD is a function of the amount of sample used [26]. In all cases, 50 lL of plasma was used for the LOD experiments. The LODs were determined using the polyclonal anti-AngI antibody obtained from the department of Pathology and Laboratory Medicine, St. Paul’s Hospital, (Vancouver, BC, Canada) and the polyclonal anti-AngII antibody obtained from BACHEM. The stable-isotope versions of AngI [(M+H)+ = 1306.6] and AngII [(M+H)+ = 1056.5] were tested and were found to exhibit affinities for the antibodies equivalent to the natural forms of the peptides. Because of this, the detection limit of the SIS peptide can be used to determine the LOD of the corresponding endogenous forms of these peptides. To determine the LOD, the duplexed iMALDI protocol was performed by spiking in decreasing concentrations of the stable isotope versions of both peptides into whole human plasma and incubating the samples for 2 h at 4 °C to allow peptide capture by the antibodies. The affinity-purified polyclonal antibody against angiotensin I was tested down to a concentration of 10 amol/lL for the stable isotope-labeled version of AngI (Fig. 2a). The LOD for this iMALDI assay can detect and quantify AngI and AngII in 50 lL of human plasma at a detection limit of 10 amol/lL (13 pg/mL) with a S/N of 2:1. This LOD is within the relevant range of patient samples. It should be noted that only one-tenth of the antibody beads were spotted onto the MALDI target in these experiments. We are therefore confident that this assay will be able to detect plasma AngI and AngII levels in normotensive patients, where the levels are 19 amol/lL (25 and 21 pg/mL, respectively [27]). Tandem MS was performed to confirm the identity of the stable isotope-labeled isoform of AngI (Fig. 2a). It should be noted that a trace of residual AngI and AngII remained bound to the anti-AngI and anti-AngII antibody during the purification process, so these detection limits were determined using the SIS peptides. If a cleaner Ab preparation were available (free of the natural form of AngI), this would enable the detection of endogenous AngI and AngII with
the same detection limits, based on the similar affinities of the corresponding SIS peptides. The affinity-purified polyclonal antiserum against angiotensin II, purchased from BACHEM, was tested for its LOD in a similar fashion. The lowest detectible concentration of the stable isotope version of AngII was 10 amol/lL (11 pg/mL), with a S/N of 2, indicating that the LOD for AngII in these experiments was 10 amol/lL (11 pg/mL) (Fig. 2b). Tandem MS was performed to confirm the identity of the stable isotope-labeled isoform of AngII (Fig. 2b). Many of the ion signals present in these LOD-level iMALDI spectra are due to non-specific binding or contamination from the beads, but these peaks do not interfere with either the endogenous or SIS peptides. Large peaks in the 99-h incubated sample at m/z 1060.2, 1098.5, and 1211.5 (data not shown) were analyzed by MS/MS and were identified as bradykinin at m/z 1060.2, and complement protein C3 fragments at m/z 1098.5 and 1211.5. This demonstrates both the specificity of iMALDI and the capability of identifying non-specifically bound peptides. While the time-zero endogenous levels of AngI were above the levels of the residual peptides resulting from the purification process, in some time-zero patient samples we were unable to detect an increase in the AngII peak ratio beyond that found for the anti-AngII beads in PBS. However, this small amount of residual AngI and AngII does not significantly affect the PRA determination, since it is a difference measurement. Given the low LOD for AngI and AngII, in samples where the level of AngI and/or AngII falls below the LOD, it is likely that only a short incubation of the plasma at 37 °C would be required to increase the amount of peptide to a detectable level. 3.2. Quantitation of AngI and AngII in plasma We next determined the linearity of the assay, and found a strong linear correlation between the amount of native angiotensin peptides and an increase in the peak area ratio between the natural and SIS forms of the peptides (Fig. 3a and b, and Supplementary Data Table I). The correlation coefficient for AngI was determined to be 0.993 (Fig. 3a); the correlation coefficient for AngII was determined to be 0.997 (Fig. 3b), over the concentration range from 4 to 100 fmol/lL (5.2–130 ng/mL) for AngI and 100–2500 amol/lL (106–2.6 ng/mL) for AngII.
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3.3. Detection of peptides in plasma Detection of both AngI and AngII from plasma with no peptide generation period was possible for each of the three plasma standards. In Fig. 4a and b, it can be seen that the peak area ratio (natural peptide: stable isotope) is greater for both AngI and AngII for each experimental sample when compared to the phosphate buffered saline (PBS) sample. The peaks corresponding to the natural forms of AngI and AngII in the PBS spectrum are attributed to a trace of the Ang peptides that were used during the antibody purification process. Thus, the antiserum itself contains low-levels of the natural form of the peptide which it is designed to target. These levels are, however, not high enough to interfere with this assay. In Fig. 5a and b there is a distinct increase in the peak area ratio across the three standard samples. The peak area ratios for AngI and AngII were noticeably greater in the plasma samples compared to the buffer (PBS) sample, indicating that endogenous AngI and AngII peptides were enriched from the plasma by the antibodies on the beads (Fig. 5a). The AngI peak area ratio values show a strong correlation with increasing plasma renin activities (PRA)
(a)
3.4. Determination of plasma renin and ACE activities The increase in the peak area ratios corresponds to an increase in the amount of endogenous peptide in the plasma. These ratios are plotted in Fig. 5c and d; the source data is shown in Supplementary Data Table II. The CVs determined by measuring the same sample in triplicate on different spots on the MALDI plate are also shown on this figure, giving the technical variability of the mass spectrometric portion of the assay. The CVs are <10% demonstrating high reproducibility, as required for clinical assays. While this is a preliminary experiment which was done without protease inhibitors to minimize non-specific cleavage/degradation
(b)
1.2 Peak Area Ratio
L3
Change in Peak Area Ratio
1 Peak Area Ratios (Natural : SIS)
values for the three standards. Likewise, the change in peak area ratios over time also correlates strongly with the PRA values (Fig. 5b). This indicates that the values produced by the mass spectrometricdetection iMALDI method are in agreement with the results obtained from the standard clinical PRA assay, and support the possibility of using the iMALDI approach to determine PRA in a clinical setting.
0.8
0.6
0.4
L2 L3
0.2
L1 L2 L1
0 0
5
10
15
20
PRA (ng/mL/hr)
Fig. 5. A comparison of the results from the iMALDI assay for angiotensin with those for plasma renin activity as determined by the DiaSorin RENCTK assay. The three data points correspond to Lyphochek Hypertension Markers Control Levels 1, 2 and 3 (BIORAD). The plasma renin activity values were obtained from the BIORAD Lyphochek instruction booklet, and were listed as of 1.70–3.30, 3.00–5.60, and 10.7–21.7 ng/mL/h, respectively. (a) The peak area ratio values were determined using the duplexed iMALDI approach for AngI and AngII, incubating the samples for 2 h at 4 °C to allow peptide capture by the antibody-coated beads. The change in peak area ratio values was determined as above, but with a 99-h incubation at 37 °C to promote an increase in renin activity and production of AngI Note that the BIORAD samples with the higher PRA values correlated to a greater change in peak area ratio. (b) The increase in the AngII peak area ratio over time (renin activity), and (c) The increase in AngI peak area ratio over time (ACE activity) using the Lyphochek Hypertension Markers Control Levels 1, 2 and 3 (BIORAD). The change in peak area ratio value was determined using the duplexed iMALDI approach for AngI and AngII. Three replicates were performed, and the CVs were in the range of 0.3–4% for all of these assays. Source data is shown in Supplementary Data Table II.
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of AngI and II, it is clear from the shape of the curves in Fig. 5c and d that renin and ACE are actively producing AngI and AngII, respectively, when incubated at 37 °C. Note the lack of increase in peak area ratio for the Level 3 sample incubated at 4 °C, demonstrating the requirement of physiological conditions (37 °C) in order for proper functioning of renin and ACE. The linear increase in AngI and AngII over the 99-h incubation is due to the abundance of substrate within the BIORAD standards. Other data (not shown) suggest that if the incubation is carried out long enough, renin substrate will ultimately become depleted, resulting in a plateau followed shortly thereafter by a depletion of ACE substrate (AngI). Some evidence of substrate depletion can already be seen in the Level 3 samples in Fig. 5b and c at the longest timepoints. (Fig. 6 and Supplementary Data Table III) were generated from a 99-h incubation of whole human plasma and shows the change in the levels of angiotensin I and II that follows the known pathway of the renin-angiotensin system. Initially, the rate of increase of AngI is much greater than that of AngII. This may be due to the availability of substrate in the plasma. The generation of AngI by renin provides more substrate for ACE, from which AngII can be generated. The plateau for both curves indicates at what point substrate depletion occurred. The decrease in AngI at the 48 and 99 h mark without an associated increase in AngII is likely due to the effects of non-specific degradation of AngI and AngII by plasma peptidases. AngII can also be cleaved by angiotensinases in the blood to produce AngIII and IV. AngII and IV have similar functions as AngII, but are less active. They are not generally reported or measured and are not associated with specific pathologies. The duplex iMALDI method, presented in this paper, is effective at detecting AngI and AngII and changes in their abundance within a given plasma sample, showing that both renin and ACE are active. 4. Conclusions The limits of detection of this iMALDI assay have been found to be 10 amol/lL for both AngI and AngII (13 and 11 pg/mL, respectively), with currently-available antibodies and instrumentation. Using this duplexed iMALDI approach on whole human plasma samples and lyophilized plasma standards with known PRAs, we were able to detect the protonated molecular ions of AngI and AngII and to accurately quantitate changes in these levels, which allowed us to monitor the relative activity of renin and ACE during plasma incubation. The calibration curves show that the assay is linear from 4 to 100 fmol/lL (5.23–130 ng/mL) for AngI and from
250
Concentration (ng/mL)
200
150
100
50
0 10
20
30
40 50 60 70 Incubation Time (h)
80
90
100 to 2500 amol/lL (0.106–2.64 ng/mL) for AngII with correlation coefficients of 0.99 for both compounds. These results illustrate the ability of the duplexed iMALDI to detect initial AngI and AngII concentrations that can be related to PRA, and likely ACE, activities. The direct, simultaneous detection of the peptides can provide clinicians with a faster, more reliable assay for insight into the current state of the RAS. In addition, the duplex iMALDI incubation of plasma is a simple, reliable method for tracking changes in Ang peptide levels, providing insight into the enzymatic activity of renin and ACE and has possible utility in stratification of patient groups. While in a normal functioning pathway, there is a direct, positive correlation between the levels of AngI and AngII, there are a number of known pathologies where ACE is over expressed independent of renin (sarcoidosis, cancers), resulting in a disproportionately high level of AngII. This duplex iMALDI assay may also have a place in tracking the effectiveness of drug treatment. When inhibitors to renin or ACE are applied, a distinct incubation curve is expected, with either AngI or both AngI and AngII levels remaining near the baseline throughout the incubation. The duplexed iMALDI assay may be a more effective and efficient way of presenting the state of the RAS as a clinically-significant measure by demonstrating the activity of both renin and ACE simultaneously and the resultant levels of AngI and AngII. The speed, accuracy, and precision of this duplexed iMALDI method indicates that it should be able to assist in determining treatment strategies on a patient-by-patient basis. Acknowledgements Borchers, C.H. Methods of Quantitation and Identification of Peptides and Proteins. Canadian Patent Application No. 2507,864, filing date 2003-12-02, awarded Oct 11, 2011 (http://brevets-patents.ic.gc.ca/opic-cipo/cpd/eng/patent/2507864/summary.html# Details). Borchers, C. Methods of Quantitation and Identification of Peptides and Proteins. A patent for iMALDI technology was been submitted by the University of North Carolina. US patent application number 7846748 B2, filing Date April 12, 2003, Awarded Dec. 7, 2010 (http://ip.com/patent/US7846748). Funds for this research were generously provided by a platform grant from Genome Canada and Genome British Columbia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymeth.2012.02.006. References
AngI AngII
0
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100
Fig. 6. Plasma renin and ACE activity. The increase in concentration corresponds to an increase in the amount of endogenous peptide in the plasma. The human plasma sample was incubated at 37 °C without any enzymatic inhibitors. The CVs were determined by measuring the same sample in triplicate on different spots on the MALDI plate. Source data is shown in Supplementary Data Table III.
[1] C.F. Deschepper, Kidney International 46 (1994) 1561–1563. [2] J. Hall, Molecular Biotechnology 24 (2003) 27–39. [3] E. Hackenthal, M. Paul, D. Ganten, R. Taugner, Physiological Reviews 70 (1990) 1067–1116. [4] P.S. Leung, Frontiers in research of the renin-angiotensin system on human disease, in: Proteases in Biology and Disease, Springer, The Netherlands, 2008, p. 308. [5] J.H.M. Van Esch, A.H.J. Danser, Local Angiotensin Generation and AT2 Receptor Activation, in: P.S. Leung (Ed.), Frontiers in Research of the Renin-Angiotensin System on Human Disease, Springer, The Netherlands, 2008, pp. 247–272. [6] M. Paul, A. Poyan Mehr, R. Kreutz, Physiological Reviews 86 (2006) 747–803. [7] M.G. Nicholls, A.M. Richards, M. Agarwal, Journal of Human Hypertension 12 (1998) 295–299. [8] L. Juillerat-Jeanneret, The Renin-Angiotensin System and its Inhibitors in Human Cancers, in: P.S. Leung (Ed.), Frontiers in Research of the ReninAngiotensin System on Human Disease, Springer, The Netherlands, 2008, pp. 197–220. [9] S. Takeda, N. Sato, T. Ogihara, R. Morishita, Frontiers in Bioscience 13 (2008) 2253–2265. [10] WHO_Cardiovascular_diseases. Available from: (accessed 26.03.10). [11] NHLBI_Fact_Book, Disease Statistics, 2008. Available from: (accessed 26.03.10).
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[12] C.E. Bystrom, W. Salameh, R. Reitz, N.J. Clarke, Clinical Chemistry 56 (2010) 1561–1569. [13] K. Poulsen, J. Jorgensen, Journal of Clinical Endocrinology and Metabolism 39 (1974) 816–825. [14] F. Fyhrquist, O. Saijonmaa, Clinical Chemistry 54 (2008) 1400. [15] E.N. Warren, P.J. Elms, C.E. Parker, C.H. Borchers, Analytical Chemistry 76 (2004) 4082–4092. [16] J. Jiang, C.E. Parker, K.A. Hoadley, C.M. Perou, G. Boysen, C.H. Borchers, Proteomics: Clinical Applications 1 (2007) 1651–1659. [17] J. Jiang, C.E. Parker, J.R. Fuller, T.H. Kawula, C.H. Borchers, Analytica Chimica Acta 605 (2007) 70–79. [18] J.D. Reid, D.T. Holmes, D.R. Mason, B. Shah, C.H. Borchers, Journal of the American Society for Mass Spectrometry 21 (2010) 1680–1686. [19] B. Shah, J.D. Reid, M.J. Kuzyk, C.E. Parker, C.H. Borchers, Methods in Molecular Biology (2012), in press.
[20] N.L. Anderson, N.G. Anderson, L.R. Haines, D.B. Hardie, R.W. Olafson, T.W. Pearson (SISCAPA), Journal of Proteome Research 3 (2004) 235–244. [21] G.L.J. Wright, L.H. Cazares, S.M. Leung, S. Nasim, B.L. Adam, T.T. Yip, P.F. Schellhammer, L. Gong, A. Vlahou, Prostate Cancer Prostatic Disease 2 (1999) 264–276. [22] T.L. Goodfriend, L. Levine, G.D. Fasman, Science 144 (1964) 1344–1346. [23] N.K. Bordeerat, N.I. Georgieva, D.G. Klapper, L.B. Collins, T.J. Cross, C.H. Borchers, J.A. Swenberg, G. Boysen, Proteomics 9 (2009) 3939–3944. [24] N. Tietz, in: A.H.B. Wu (Ed.), Tietz Clinical Guide to Laboratory Tests, fourth ed., Elsevier Health Sciences, St. Louis, Mo, 2006, p. 948. [25] D.C. Harris, Quantitative Chemical Analysis, W.H. Fremann, New York, 1991. [26] J.R. Whiteaker, L. Zhao, L. Anderson, A.G. Paulovich, Molecular & Cellular Proteomics 9 (2010) 184–196. [27] N. Tietz, in: A.H.B. Wu (Ed.), Tietz Clinical Guide to Laboratory Tests, fourth ed., Elsevier Health Sciences, St. Louis, Mo, 2006, p. 114.
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Methods journal homepage: www.elsevier.com/locate/ymeth
Duplexed iMALDI for the detection of angiotensin I and angiotensin II D. Randal Mason a, Jennifer D. Reid a, Alexander G. Camenzind a, Daniel T. Holmes b, Christoph H. Borchers a,c,⇑ a
University of Victoria-Genome British Columbia Proteomics Centre, Victoria, British Columbia, Canada V8Z 7X8 University of British Columbia, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, 1081 Burrard St., Vancouver, British Columbia, Canada V6Z 1Y6 c University of Victoria, Department of Biochemistry and Microbiology, Victoria, British Columbia, Canada V8Z 7X8 b
a r t i c l e
i n f o
Article history: Available online 25 February 2012 Keywords: Angiotensin I Angiotensin II Plasma renin activity ACE activity iMALDI Hypertension Immunoaffinity mass spectrometry Duplex iMALDI
a b s t r a c t An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a 50 lL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of 10 amol/lL (or 13 pg/mL AngI and 11 pg/ mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-lL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4–100 fmol/lL (5–130 ng/mL) and 100–2500 amol/lL (106–2614 pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction 1.1. Renin-angiotensin system Discovered over 100 years ago, the renin-angiotensin system (RAS) is a biochemical pathway which plays a critical role in cardiovascular physiology (Scheme 1) [1]. The pathway begins with angiotensinogen, an a-2 globulin constitutively manufactured by
the liver [1]. Of the 452 amino acids in its sequence, only the first 10 are known to play a role in RAS, and angiotensinogen itself has no known independent function [2]. Renin is the next critical RAS protein. Its enzymatic precursor, prorenin, is secreted into the bloodstream from the juxtaglomerular apparatus of kidneys. Once it has been activated to renin, it cleaves the decapeptide angiotensin I (AngI, DRVYIHPFHL) [3] from angiotensinogen. Like angiotensinogen, AngI itself has no known
Scheme 1. RAS overview.
Abbreviations: ACE, angiotensin-converting enzyme; AngI, angiotensin I; AngII, angiotensin II; iMALDI, immunoMALDI (immuno-Matrix-Assisted Laser Desorption/ Ionization); PRA, plasma renin activity; RAS, renin-angiotensin system; SIS, stable-isotope-labeled internal standard; LOD, Limit of Detection; S/N, signal-to-noise ratio; MS, mass spectrometry; MS/MS, tandem mass spectrometry; CV, coefficient of variation; PAR, peak area ratio. ⇑ Corresponding author. Address: University of Victoria-Genome British Columbia Proteomics Centre, 3101-4464 Markham St., Victoria, British Columbia, Canada V8Z 7X8. Fax: +1 250 483 3238. E-mail address: [email protected] (C.H. Borchers), [email protected] (C.H. Borchers). 1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2012.02.006
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Add Labeled Angiotensin I and II Peptide Standards
Whole Plasma
Angiotensin II Angiotensin I Add Antiangiotensin Affinity Beads
MS
m/z
MS2
analyze
Angiotensin Capture Period m/z
Fig. 1. iMALDI workflow schematic.
physiological activity but is the substrate for angiotensin converting enzyme (ACE). ACE is a metalloprotease, which cleaves two Cterminal residues from AngI to produce angiotensin II (AngII, DRVYIHPF), the primary physiological effector of the RAS pathway, acting as an endocrine, paracrine/autocrine and intracrine hormone [4]. In recent years, a number of different Ang receptors, expressed by different tissues, have been identified for which AngII is a ligand [5]. Binding of the AngII ligand to these receptors increases blood pressure directly by vasoconstriction and indirectly by stimulating the manufacture and release of aldosterone, anti-diuretic hormone (ADH) and through stimulation of the sympathetic nervous system [6].
1.2. RAS and disease states Physiological derangements of the RAS may arise from within the system itself, for example, through acquired or inherited mutation leading to alteration in the function and/or abundance of its various components. An imbalance in RAS may also be a response to changes in related physiological systems such as mechanical stress to the body, environmental or dietary changes or some other primary disease [4]. Traditionally, pathological upregulation of RAS (e.g., from renal arterial stenosis, primary renal parenchymal diseases, estrogen effect) has been associated primarily with cardiovascular risk due to the hypertensive effects of AngII [7]. More recently, the RAS has been implicated in a wide range of conditions from musculoskeletal diseases, to Alzheimer’s to various cancers. In cancers, changes in RAS activity are associated with tumor angiogenesis, which is required for continued tumor growth. RAS enzyme inhibitors have been shown to have anti-tumor effects [8]. With respect to Alzheimer’s disease, there is an epidemiological correlation between hypertension and the development of this disease [9]. It has also been recently discovered that the brain itself has a local RAS that may mediate the development of Alzheimer’s disease [6,9]. Cardiovascular diseases are the number one killer globally, with an annual combined (direct and indirect) treatment cost that exceeds $430 billion dollars
[10,11]. Therefore, because of its association with all of these diseases, the biochemical investigation and therapeutic regulation of RAS is of crucial importance to public health. 1.3. Methods of assessing RAS The RAS is routinely monitored by measuring the concentrations of AngI or AngII, renin or ACE using competitive radioimmunoassay and ELISA. Mass spectrometry methods have also been developed for AngI and AngII, including an LC-MRM method [12]. Immunoradiometric and chemiluminometric assays are available for measuring the concentration of renin in plasma. Spectrophotometric assays are also used for determining ACE activity, where a synthetic substrate is used for the measurement. Functional enzymatic assays are also available, which determine the ability of the RAS system to regulate arterial pressure by measuring the enzymatic activity of renin or ACE. The low levels of Ang peptides in plasma has favored the utilization of functional enzymatic assays, whereby a standardized incubation period is used to increase the levels of the Ang peptides above those that would occur endogenously in order to enable their detection. The plasma renin activity is the most common method of assessing RAS function and measures the production of AngI as a product of angiotensinogen cleavage by renin. As biochemical methods have evolved, the assessment of renin activity moved from bioassay methods to the radioimmunoassay methods that are still commonly in use today [13,14]. 1.4. The iMALDI method iMALDI is a technique that involves affinity capture of the peptide of interest on anti-peptide Ab-coated beads, which are washed to remove non-specifically-bound interferences (Fig. 1) [15–19]. After washing, the beads are spotted directly onto a MALDI target plate for mass spectrometric analysis. The beads remain on the target. There is no prior elution of the analyte – the analyte is eluted by the matrix solution. The principle of immunocapture is also used in SISCAPA (Stable Isotope Standards and Capture by Anti-
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Peptide Antibodies, [20]) and in some versions of SELDI (SurfaceEnhanced Laser Desorption/Ionization [21]). SISCAPA and iMALDI both use anti-peptide antibodies for affinity purification and cocapture of stable-isotope-labeled standards for quantitation. However, in iMALDI, the analytes are not eluted from the beads and MALDI is used for the analysis. The SELDI approach relies on antibodies (not necessarily anti-peptide antibodies) but in SELDI, the surface of the MALDI target is modified with these capture agents. iMALDI has the advantage of using standard MALDI targets, and varying amounts of affinity beads can be placed on the target used to produce the required detection sensitivity. Multiple iMALDI assays, with different target peptides, can be done on a single target, leading to a very flexible analytical platform, with MALDI-MS/MS providing the needed specificity. Another advantage of iMALDI is that it is a simple methodology that lends well to automation, producing assays that are inexpensive, sensitive, and specific. Due to the unique masses and sequences of the target peptides, mass spectrometric analysis provides attomole-level LODs on-target (using only 50 lL of plasma) and with very high, if not absolute, specificity of detection. In addition, once the bead-Ab-peptide conjugates are spotted on the target, the analyte can remain stable for months and be reanalyzed if necessary. The iMALDI technique has proven to be effective in detecting: low-abundance bacterial proteins in human plasma [17], the presence of upregulated receptors in tumorigenic cells [16], and the detection of AngI alone in human plasma [18]. iMALDI also allows for the simultaneous detection (multiplexing) of multiple peptides from a common complex background.
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Synthetic AngI and AngII were purchased from Sigma–Aldrich. Isotopically labeled AngI and AngII were synthesized with a stable-isotope-coded arginine residue (+10 Da) (DRVYIHPFHL and DRVYIHPF, respectively) at the University of Victoria Genome BC Proteomics Centre (Victoria, BC, Canada), according to previouslydescribed protocols [23]. 2.2. Antibody-bead conjugation Magnetic Protein G Dynabeads were precipitated in the manufacturer’s detergent-containing storage buffer using a DynaMag-2 magnet (Invitrogen). The binding capacity of the Protein G Dynabeads was determined according to the manufacturer’s specifications, and an excess of beads was used in these iMALDI experiments to ensure maximum binding of the antibody. The supernatant was removed, and the beads were washed three times in 50 lL aliquots of 1 phosphate buffered saline (PBS) by vortexing the beads in buffer and removing the supernatant in order to discard as much of the storage buffer as possible. A 5 lL-aliquot of beads was used for each reaction. Antibodies (2 lL anti-AngI pAb or 1 lL anti-AngII pAb) were immobilized on Protein G Dynabeads in 1 PBS, pH 7.4 for 1 h at 4 °C. Following the 1-h incubation, excess antibody was removed by washing the beads three times in 1 PBS (50 lL). Beads were resuspended in 1 PBS (10 lL buffer per reaction) and then aliquoted into the individual samples. For the duplexed experiments, the anti-AngI pAb-bead conjugates and anti-AngII pAb-bead conjugates were combined at this stage. 2.3. PRA standards
1.5. Advantages of a duplexed iMALDI assay With regard to the RAS, we hypothesized that it should be possible to detect the presence and levels of both AngI and AngII in whole human plasma. The molecular weights of AngI and AngII are well-suited to iMALDI, (MW’s of 1295.6 Da and 1045.5 Da, respectively), which allows these peptides to be detected from plasma without the need for tryptic digestion, which is required in most proteomics experiments, and is normally included in the standard iMALDI workflow. With a duplexed iMALDI assay, it is possible to simultaneously quantify both AngI and AngII, in a single experiment, in a single sample. Because this is a molecular weight-based assay with mass spectrometric detection, there is no crossreactivity between AngI and AngII. The ability to simultaneously measure the levels of these two peptides may also aid in the monitoring of these different pathologies. Thus with the duplexed iMALDI approach, it is possible to simultaneously quantify AngI, AngII and, with incubation steps, renin and ACE activities also. 2. Methods 2.1. Chemicals and reagents Polyclonal antiserum (pAb) to AngI was obtained by immunizing rabbits with angiotensin-I/albumin complexes in Freund’s adjuvant [22]. Anti-AngII pAb (2 mg/mL) was purchased from BACHEM (Torrance, CA). The polyclonal anti-AngI antibody and the human plasma samples were obtained from the department of Pathology and Laboratory Medicine, St. Paul’s Hospital (Vancouver, BC, Canada). The anti-AngI pAb and anti-AngII pAb were both affinity purified antisera and as such, the antisera itself contains bound AngI and AngII peptides, respectively. Antibody-bead conjugation was performed using magnetic Protein G Dynabeads (30 mg/mL) (Invitrogen).
The samples used for these experiments were Lyphochek™ Hypertension Markers Control Levels 1, 2 and 3 from BIO-RAD (lot number 35940, Irvine, CA)., which consist of lyophilized human plasma. The BIO-RAD samples consist of processed lyophilized human plasma with added constituents of human and animal origin. They are used as quality-control markers for hypertension assays such as the DiaSorin Gamma Coat assay for plasma renin activity (DiaSorin Canada, Mississauga, ON, P2621). The standards have known DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60 and 10.7–21.7 ng/mL/h for the Level 1, Level 2, and Level 3 standards, respectively. The normal range for plasma renin activity is 0.73–3.96 ng/mL/h [24] which would place the Levels 1, 2, and 3 standards from BIO-RAD at the normal, high-normal, and high range for plasma renin activity, respectively. 2.4. MALDI instrument parameters MALDI MS and MS/MS data were acquired on an AB Sciex 4800 MALDI mass spectrometer. The instrument is equipped with a solid-state ND:YAG laser, operated at 200 Hz. The source was operated in the positive ion reflectron mode. The MS/MS setting was 2KV (positive). An average of 100 shots/sub-spectrum was acquired in the MS mode, with a total of 1000 shots per spectrum. In the MS/MS mode, 25 shots were acquired per subspectrum, with a total of 40 sub-spectra acquired and a total of 1000 total shots per spectrum. Delayed extraction was on, and the MS/MS fragmentation mechanism was CID. Applied Biosystems 4000-Series Explorer software was used for instrument control; Applied Biosystems Data Explorer was used for the Data Analysis. Peak areas were used for quantitation. 2.5. Duplexed iMALDI Following antibody-bead conjugation, the PBS supernatant was removed using the magnet and the beads were resuspended in 50 lL of either PBS or whole human plasma. The stable-isotope
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Table 1 The concentrations of stable isotope peptides spiked into 50 lL of whole human plasma to determine the Limit of Detection of the duplexed iMALDI assay. Sample
[AngI] amol/lL
[AngI] ng/mL
[AngII] amol/lL
[AngII] ng/mL
1 2 3 4 5
500 100 50 25 10
0.653 0.131 0.065 0.033 0.013
500 100 50 25 10
0.528 0.106 0.053 0.026 0.011
versions of AngI and AngII were added at known amounts to the samples to act as a reference standards. The samples were then incubated at 37 °C (4 °C for the control sample), where endogenous AngI and AngII and the internal standards were affinity-captured from the plasma solution by the antibody-bead conjugates. Following incubation, the beads were separated using the DynaMag-2 magnet, the supernatant was removed and the beads were washed four times in 25 mM ammonium bicarbonate (ABC). The samples were then re-suspended in 10 lL 25 mM ABC for the duplexed
Fig. 2. iMALDI mass spectra of 25 amol/lL AngI and AngII captured on affinity beads (a) MALDI-MS/MS and MALDI-MS spectra of AngI SIS (b) MALDI-MS/MS and MALDI-MS spectra of AngII SIS.
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Fig. 3. Linearity of the duplexed iMALDI assay as demonstrated by the addition of varying levels of the SIS peptides to a human plasma sample. (a) Heavy:light peptide peak area ratios for different levels of AngI (b) Heavy:light peptide peak area ratios for different levels of AngII. Source data is shown in Supplementary Data Table I.
samples (5lL for non-duplexed samples). A 1-lL aliquot of each sample was then spotted in triplicate onto a polished steel, 384-well MALDI plate. Once dry, each sample was coated with 1 lL of CHCA matrix (10 mM a-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% trifluoroacetic acid). The samples were then analyzed using an Applied Biosystems 4800 MALDI TOF/TOF mass spectrometer. 2.6. Assay validation 2.6.1. Linearity and quantitation Determination of the linearity of the iMALDI assay and quantitation were performed by adding standard amounts of the synthetic natural forms of AngI (20 fmol/lL; 26 ng/mL) and AngII (0.5 fmol/lL; 0.53 ng/mL), while varying the amount of the stable-isotope versions of AngI (0–100 fmol/lL; 0–130 ng/mL) and AngII (0–2.5 fmol/lL; 0–2.6 ng/mL) added to PBS buffer and whole human plasma. Using the standard-addition method, this generated a regression line from which the endogenous levels of AngI and AngII could be assessed [25]. The peak area ratios (PARs) of the natural and stable isotope forms of the peptide were used to quantify the natural form of the peptide and to detect an increase in endogenous levels over time when a known amount of stable isotope was introduced to the sample. 2.6.2. Limit of Detection for duplexed iMALDI The Limit of Detection (LOD) was determined by spiking known concentrations of the stable-isotope versions of AngI (m/z 1306.6) and AngII (m/z 1056.5) into a pooled whole human plasma sample. We had previously established that the stable isotope and natural forms of the peptides demonstrated equal affinities for their respective antibodies (data not shown). The duplexed approach described above was employed, with stable isotope standards of AngI and AngII being spiked into different plasma samples at the concentrations shown in Table 1. The samples were incubated for 2 h at 4 °C, then washed and analyzed via MALDI-TOF/TOF mass spectrometry. The LOD for the duplexed iMALDI assay was defined as the lowest concentration at which the peak associated with the SIS form of the Ang peptide could consistently be detected at a S/N of 2:1. Tandem mass spectrometric sequencing was performed on the samples containing the lowest detectable peaks to confirm the identities of the given peptides.
2.6.3. Detection and time course studies of AngI and AngII Lyphochek™ Hypertension Controls (Levels 1, 2, and 3, corresponding to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60, and 10.7–21.7 ng/mL/h) were used to track changes in the concentrations of AngI and AngII over time. This experiment was carried out without the addition of any enzymatic inhibitors, using 350 lL of plasma and stable isotopes at concentrations of 20 fmol/lL (26 ng/mL) and 500 amol/lL (0.53 ng/mL) for AngI and AngII, respectively, and the equivalent of seven 10-lL aliquots of antibody-bead conjugates. Following the incubation of plasma with the stable-isotope standards of AngI and AngII and antibody-bead conjugates for 3 h at 4 °C (to allow peptide binding), the samples were incubated at 37 °C to promote AngI and AngII generation. Fifty-microliters aliquots were taken from the sample at the following time points: 0, 4, 7, 12, 24, 48, and 99 h, the beads were washed, re-suspended, and analyzed as described above. The technical variability of the mass spectrometric portion of the assay was assessed by spotting the same sample in triplicate on the MALDI target plate. Each sample spot was measured five times, and the intra-spot coefficient of variation (CVs) and standard deviation were determined. The average of the five acquisitions is then used as a representative value for that spot, and the CV was then taken across three replicate sample spots. The LOD results were taken from the 0-h time point of this plasma incubation experiment where, with a 2-h incubation at 4 °C, there would have been a very low level of enzymatic activity in the plasma. 3. Results and discussion We have previously developed an iMALDI assay for AngI in human plasma [16]. This iMALDI approach also provided an improved LOD of synthetic angiotensin I in buffer by 80-fold compared to AngI spotted directly on a MALDI target. The LOD for AngI in these experiments was determined to be 65 pg/mL, thus meeting the clinical requirements for measuring low renin activity. Moreover, because of its high sensitivity, only 50 lL of plasma is needed for the iMALDI assay which is lower than that required for many automated immunoassay systems for various human proteins. Because of the sensitivity of this assay and the specificity of iMALDI, we hypothesized that it should be possible to develop an assay for the quantitation of both AngI and AngII, simultaneously. After incubation, the assay would also be valuable in identifying any perturbations in renin and/or ACE activity that might assist the clinician identifying a specific pathology.
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Fig. 4. (a) iMALDI spectrum showing both AngI and AngII, endogenous and SIS forms. (b) Detection of AngI in whole human plasma using duplexed iMALDI. The spectra labeled ‘‘Level 1’’, ‘‘Level 2’’, and ‘‘Level 3’’ are from three BIORAD standards (which correspond to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60, and 10.7– 21.7 ng/mL/h, respectively), while spectrum labeled ‘‘buffer’’ is the result of incubating the antibody beads in phosphate buffered saline (PBS); (c) detection of angiotensin II (AngII) in whole human plasma using the duplexed iMALDI, labeled as above; (d) simultaneous detection and relative quantitation of angiotensins I and II in BIO-RAD hypertension plasma standards through the duplexed iMALDI. The samples were incubated in 50 lL of plasma for 2 h at 4 °C to allow antibody capture of the peptides. The error bars reflect the CV determined by measuring the same sample in triplicate on different spots on the MALDI plate (L1, Level 1; L2, Level 2; and L3, Level 3; corresponding to DiaSorin Gamma Coat value ranges of 1.70–3.30, 3.00–5.60 and 10.7–21.7 ng/mL/h, respectively).
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Fig. 4 (continued)
3.1. Determination of the LOD The Limit of Detection (LOD) of any iMALDI assay, including this duplexed assay, is highly dependent on the affinities of the antibodies used for the target peptides. LODs for this assay were defined as the lowest analyte concentration which could reliably be distinguished from background and could be confirmed by tandem MS/MS with a S/N cutoff of 2:1. As with other affinity-based techniques, such as SISCAPA, the LOD is a function of the amount of sample used [26]. In all cases, 50 lL of plasma was used for the LOD experiments. The LODs were determined using the polyclonal anti-AngI antibody obtained from the department of Pathology and Laboratory Medicine, St. Paul’s Hospital, (Vancouver, BC, Canada) and the polyclonal anti-AngII antibody obtained from BACHEM. The stable-isotope versions of AngI [(M+H)+ = 1306.6] and AngII [(M+H)+ = 1056.5] were tested and were found to exhibit affinities for the antibodies equivalent to the natural forms of the peptides. Because of this, the detection limit of the SIS peptide can be used to determine the LOD of the corresponding endogenous forms of these peptides. To determine the LOD, the duplexed iMALDI protocol was performed by spiking in decreasing concentrations of the stable isotope versions of both peptides into whole human plasma and incubating the samples for 2 h at 4 °C to allow peptide capture by the antibodies. The affinity-purified polyclonal antibody against angiotensin I was tested down to a concentration of 10 amol/lL for the stable isotope-labeled version of AngI (Fig. 2a). The LOD for this iMALDI assay can detect and quantify AngI and AngII in 50 lL of human plasma at a detection limit of 10 amol/lL (13 pg/mL) with a S/N of 2:1. This LOD is within the relevant range of patient samples. It should be noted that only one-tenth of the antibody beads were spotted onto the MALDI target in these experiments. We are therefore confident that this assay will be able to detect plasma AngI and AngII levels in normotensive patients, where the levels are 19 amol/lL (25 and 21 pg/mL, respectively [27]). Tandem MS was performed to confirm the identity of the stable isotope-labeled isoform of AngI (Fig. 2a). It should be noted that a trace of residual AngI and AngII remained bound to the anti-AngI and anti-AngII antibody during the purification process, so these detection limits were determined using the SIS peptides. If a cleaner Ab preparation were available (free of the natural form of AngI), this would enable the detection of endogenous AngI and AngII with
the same detection limits, based on the similar affinities of the corresponding SIS peptides. The affinity-purified polyclonal antiserum against angiotensin II, purchased from BACHEM, was tested for its LOD in a similar fashion. The lowest detectible concentration of the stable isotope version of AngII was 10 amol/lL (11 pg/mL), with a S/N of 2, indicating that the LOD for AngII in these experiments was 10 amol/lL (11 pg/mL) (Fig. 2b). Tandem MS was performed to confirm the identity of the stable isotope-labeled isoform of AngII (Fig. 2b). Many of the ion signals present in these LOD-level iMALDI spectra are due to non-specific binding or contamination from the beads, but these peaks do not interfere with either the endogenous or SIS peptides. Large peaks in the 99-h incubated sample at m/z 1060.2, 1098.5, and 1211.5 (data not shown) were analyzed by MS/MS and were identified as bradykinin at m/z 1060.2, and complement protein C3 fragments at m/z 1098.5 and 1211.5. This demonstrates both the specificity of iMALDI and the capability of identifying non-specifically bound peptides. While the time-zero endogenous levels of AngI were above the levels of the residual peptides resulting from the purification process, in some time-zero patient samples we were unable to detect an increase in the AngII peak ratio beyond that found for the anti-AngII beads in PBS. However, this small amount of residual AngI and AngII does not significantly affect the PRA determination, since it is a difference measurement. Given the low LOD for AngI and AngII, in samples where the level of AngI and/or AngII falls below the LOD, it is likely that only a short incubation of the plasma at 37 °C would be required to increase the amount of peptide to a detectable level. 3.2. Quantitation of AngI and AngII in plasma We next determined the linearity of the assay, and found a strong linear correlation between the amount of native angiotensin peptides and an increase in the peak area ratio between the natural and SIS forms of the peptides (Fig. 3a and b, and Supplementary Data Table I). The correlation coefficient for AngI was determined to be 0.993 (Fig. 3a); the correlation coefficient for AngII was determined to be 0.997 (Fig. 3b), over the concentration range from 4 to 100 fmol/lL (5.2–130 ng/mL) for AngI and 100–2500 amol/lL (106–2.6 ng/mL) for AngII.
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3.3. Detection of peptides in plasma Detection of both AngI and AngII from plasma with no peptide generation period was possible for each of the three plasma standards. In Fig. 4a and b, it can be seen that the peak area ratio (natural peptide: stable isotope) is greater for both AngI and AngII for each experimental sample when compared to the phosphate buffered saline (PBS) sample. The peaks corresponding to the natural forms of AngI and AngII in the PBS spectrum are attributed to a trace of the Ang peptides that were used during the antibody purification process. Thus, the antiserum itself contains low-levels of the natural form of the peptide which it is designed to target. These levels are, however, not high enough to interfere with this assay. In Fig. 5a and b there is a distinct increase in the peak area ratio across the three standard samples. The peak area ratios for AngI and AngII were noticeably greater in the plasma samples compared to the buffer (PBS) sample, indicating that endogenous AngI and AngII peptides were enriched from the plasma by the antibodies on the beads (Fig. 5a). The AngI peak area ratio values show a strong correlation with increasing plasma renin activities (PRA)
(a)
3.4. Determination of plasma renin and ACE activities The increase in the peak area ratios corresponds to an increase in the amount of endogenous peptide in the plasma. These ratios are plotted in Fig. 5c and d; the source data is shown in Supplementary Data Table II. The CVs determined by measuring the same sample in triplicate on different spots on the MALDI plate are also shown on this figure, giving the technical variability of the mass spectrometric portion of the assay. The CVs are <10% demonstrating high reproducibility, as required for clinical assays. While this is a preliminary experiment which was done without protease inhibitors to minimize non-specific cleavage/degradation
(b)
1.2 Peak Area Ratio
L3
Change in Peak Area Ratio
1 Peak Area Ratios (Natural : SIS)
values for the three standards. Likewise, the change in peak area ratios over time also correlates strongly with the PRA values (Fig. 5b). This indicates that the values produced by the mass spectrometricdetection iMALDI method are in agreement with the results obtained from the standard clinical PRA assay, and support the possibility of using the iMALDI approach to determine PRA in a clinical setting.
0.8
0.6
0.4
L2 L3
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L1 L2 L1
0 0
5
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PRA (ng/mL/hr)
Fig. 5. A comparison of the results from the iMALDI assay for angiotensin with those for plasma renin activity as determined by the DiaSorin RENCTK assay. The three data points correspond to Lyphochek Hypertension Markers Control Levels 1, 2 and 3 (BIORAD). The plasma renin activity values were obtained from the BIORAD Lyphochek instruction booklet, and were listed as of 1.70–3.30, 3.00–5.60, and 10.7–21.7 ng/mL/h, respectively. (a) The peak area ratio values were determined using the duplexed iMALDI approach for AngI and AngII, incubating the samples for 2 h at 4 °C to allow peptide capture by the antibody-coated beads. The change in peak area ratio values was determined as above, but with a 99-h incubation at 37 °C to promote an increase in renin activity and production of AngI Note that the BIORAD samples with the higher PRA values correlated to a greater change in peak area ratio. (b) The increase in the AngII peak area ratio over time (renin activity), and (c) The increase in AngI peak area ratio over time (ACE activity) using the Lyphochek Hypertension Markers Control Levels 1, 2 and 3 (BIORAD). The change in peak area ratio value was determined using the duplexed iMALDI approach for AngI and AngII. Three replicates were performed, and the CVs were in the range of 0.3–4% for all of these assays. Source data is shown in Supplementary Data Table II.
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of AngI and II, it is clear from the shape of the curves in Fig. 5c and d that renin and ACE are actively producing AngI and AngII, respectively, when incubated at 37 °C. Note the lack of increase in peak area ratio for the Level 3 sample incubated at 4 °C, demonstrating the requirement of physiological conditions (37 °C) in order for proper functioning of renin and ACE. The linear increase in AngI and AngII over the 99-h incubation is due to the abundance of substrate within the BIORAD standards. Other data (not shown) suggest that if the incubation is carried out long enough, renin substrate will ultimately become depleted, resulting in a plateau followed shortly thereafter by a depletion of ACE substrate (AngI). Some evidence of substrate depletion can already be seen in the Level 3 samples in Fig. 5b and c at the longest timepoints. (Fig. 6 and Supplementary Data Table III) were generated from a 99-h incubation of whole human plasma and shows the change in the levels of angiotensin I and II that follows the known pathway of the renin-angiotensin system. Initially, the rate of increase of AngI is much greater than that of AngII. This may be due to the availability of substrate in the plasma. The generation of AngI by renin provides more substrate for ACE, from which AngII can be generated. The plateau for both curves indicates at what point substrate depletion occurred. The decrease in AngI at the 48 and 99 h mark without an associated increase in AngII is likely due to the effects of non-specific degradation of AngI and AngII by plasma peptidases. AngII can also be cleaved by angiotensinases in the blood to produce AngIII and IV. AngII and IV have similar functions as AngII, but are less active. They are not generally reported or measured and are not associated with specific pathologies. The duplex iMALDI method, presented in this paper, is effective at detecting AngI and AngII and changes in their abundance within a given plasma sample, showing that both renin and ACE are active. 4. Conclusions The limits of detection of this iMALDI assay have been found to be 10 amol/lL for both AngI and AngII (13 and 11 pg/mL, respectively), with currently-available antibodies and instrumentation. Using this duplexed iMALDI approach on whole human plasma samples and lyophilized plasma standards with known PRAs, we were able to detect the protonated molecular ions of AngI and AngII and to accurately quantitate changes in these levels, which allowed us to monitor the relative activity of renin and ACE during plasma incubation. The calibration curves show that the assay is linear from 4 to 100 fmol/lL (5.23–130 ng/mL) for AngI and from
250
Concentration (ng/mL)
200
150
100
50
0 10
20
30
40 50 60 70 Incubation Time (h)
80
90
100 to 2500 amol/lL (0.106–2.64 ng/mL) for AngII with correlation coefficients of 0.99 for both compounds. These results illustrate the ability of the duplexed iMALDI to detect initial AngI and AngII concentrations that can be related to PRA, and likely ACE, activities. The direct, simultaneous detection of the peptides can provide clinicians with a faster, more reliable assay for insight into the current state of the RAS. In addition, the duplex iMALDI incubation of plasma is a simple, reliable method for tracking changes in Ang peptide levels, providing insight into the enzymatic activity of renin and ACE and has possible utility in stratification of patient groups. While in a normal functioning pathway, there is a direct, positive correlation between the levels of AngI and AngII, there are a number of known pathologies where ACE is over expressed independent of renin (sarcoidosis, cancers), resulting in a disproportionately high level of AngII. This duplex iMALDI assay may also have a place in tracking the effectiveness of drug treatment. When inhibitors to renin or ACE are applied, a distinct incubation curve is expected, with either AngI or both AngI and AngII levels remaining near the baseline throughout the incubation. The duplexed iMALDI assay may be a more effective and efficient way of presenting the state of the RAS as a clinically-significant measure by demonstrating the activity of both renin and ACE simultaneously and the resultant levels of AngI and AngII. The speed, accuracy, and precision of this duplexed iMALDI method indicates that it should be able to assist in determining treatment strategies on a patient-by-patient basis. Acknowledgements Borchers, C.H. Methods of Quantitation and Identification of Peptides and Proteins. Canadian Patent Application No. 2507,864, filing date 2003-12-02, awarded Oct 11, 2011 (http://brevets-patents.ic.gc.ca/opic-cipo/cpd/eng/patent/2507864/summary.html# Details). Borchers, C. Methods of Quantitation and Identification of Peptides and Proteins. A patent for iMALDI technology was been submitted by the University of North Carolina. US patent application number 7846748 B2, filing Date April 12, 2003, Awarded Dec. 7, 2010 (http://ip.com/patent/US7846748). Funds for this research were generously provided by a platform grant from Genome Canada and Genome British Columbia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymeth.2012.02.006. References
AngI AngII
0
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Fig. 6. Plasma renin and ACE activity. The increase in concentration corresponds to an increase in the amount of endogenous peptide in the plasma. The human plasma sample was incubated at 37 °C without any enzymatic inhibitors. The CVs were determined by measuring the same sample in triplicate on different spots on the MALDI plate. Source data is shown in Supplementary Data Table III.
[1] C.F. Deschepper, Kidney International 46 (1994) 1561–1563. [2] J. Hall, Molecular Biotechnology 24 (2003) 27–39. [3] E. Hackenthal, M. Paul, D. Ganten, R. Taugner, Physiological Reviews 70 (1990) 1067–1116. [4] P.S. Leung, Frontiers in research of the renin-angiotensin system on human disease, in: Proteases in Biology and Disease, Springer, The Netherlands, 2008, p. 308. [5] J.H.M. Van Esch, A.H.J. Danser, Local Angiotensin Generation and AT2 Receptor Activation, in: P.S. Leung (Ed.), Frontiers in Research of the Renin-Angiotensin System on Human Disease, Springer, The Netherlands, 2008, pp. 247–272. [6] M. Paul, A. Poyan Mehr, R. Kreutz, Physiological Reviews 86 (2006) 747–803. [7] M.G. Nicholls, A.M. Richards, M. Agarwal, Journal of Human Hypertension 12 (1998) 295–299. [8] L. Juillerat-Jeanneret, The Renin-Angiotensin System and its Inhibitors in Human Cancers, in: P.S. Leung (Ed.), Frontiers in Research of the ReninAngiotensin System on Human Disease, Springer, The Netherlands, 2008, pp. 197–220. [9] S. Takeda, N. Sato, T. Ogihara, R. Morishita, Frontiers in Bioscience 13 (2008) 2253–2265. [10] WHO_Cardiovascular_diseases. Available from:
222
D.R. Mason et al. / Methods 56 (2012) 213–222
[12] C.E. Bystrom, W. Salameh, R. Reitz, N.J. Clarke, Clinical Chemistry 56 (2010) 1561–1569. [13] K. Poulsen, J. Jorgensen, Journal of Clinical Endocrinology and Metabolism 39 (1974) 816–825. [14] F. Fyhrquist, O. Saijonmaa, Clinical Chemistry 54 (2008) 1400. [15] E.N. Warren, P.J. Elms, C.E. Parker, C.H. Borchers, Analytical Chemistry 76 (2004) 4082–4092. [16] J. Jiang, C.E. Parker, K.A. Hoadley, C.M. Perou, G. Boysen, C.H. Borchers, Proteomics: Clinical Applications 1 (2007) 1651–1659. [17] J. Jiang, C.E. Parker, J.R. Fuller, T.H. Kawula, C.H. Borchers, Analytica Chimica Acta 605 (2007) 70–79. [18] J.D. Reid, D.T. Holmes, D.R. Mason, B. Shah, C.H. Borchers, Journal of the American Society for Mass Spectrometry 21 (2010) 1680–1686. [19] B. Shah, J.D. Reid, M.J. Kuzyk, C.E. Parker, C.H. Borchers, Methods in Molecular Biology (2012), in press.
[20] N.L. Anderson, N.G. Anderson, L.R. Haines, D.B. Hardie, R.W. Olafson, T.W. Pearson (SISCAPA), Journal of Proteome Research 3 (2004) 235–244. [21] G.L.J. Wright, L.H. Cazares, S.M. Leung, S. Nasim, B.L. Adam, T.T. Yip, P.F. Schellhammer, L. Gong, A. Vlahou, Prostate Cancer Prostatic Disease 2 (1999) 264–276. [22] T.L. Goodfriend, L. Levine, G.D. Fasman, Science 144 (1964) 1344–1346. [23] N.K. Bordeerat, N.I. Georgieva, D.G. Klapper, L.B. Collins, T.J. Cross, C.H. Borchers, J.A. Swenberg, G. Boysen, Proteomics 9 (2009) 3939–3944. [24] N. Tietz, in: A.H.B. Wu (Ed.), Tietz Clinical Guide to Laboratory Tests, fourth ed., Elsevier Health Sciences, St. Louis, Mo, 2006, p. 948. [25] D.C. Harris, Quantitative Chemical Analysis, W.H. Fremann, New York, 1991. [26] J.R. Whiteaker, L. Zhao, L. Anderson, A.G. Paulovich, Molecular & Cellular Proteomics 9 (2010) 184–196. [27] N. Tietz, in: A.H.B. Wu (Ed.), Tietz Clinical Guide to Laboratory Tests, fourth ed., Elsevier Health Sciences, St. Louis, Mo, 2006, p. 114.