Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma

Analytical Biochemistry 442 (2013) 1–9

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Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma Eugene Y. Zhen ⇑, Richard E. Higgs, Jesus A. Gutierrez Eli Lilly and Company, Indianapolis, IN 46285, USA

a r t i c l e

i n f o

Article history: Received 4 February 2013 Received in revised form 19 June 2013 Accepted 1 July 2013 Available online 16 July 2013 Keywords: Apelin Peptide quantification Mass spectrometry Cation exchange

a b s t r a c t Apelin is emerging as an important hormone regulator of cardiovascular homoeostasis and an important biomarker for heart failure. Apelin concentrations have historically been measured by immunoassays; however, reported apelin concentrations measured in healthy volunteers show a large disparity from a few picograms per milliliter (pg/ml) to several nanograms per milliliter (ng/ml). Apelin exists in several isoforms ranging in size from 12 to 36 residues, and immunoassays generally cannot distinguish the specific forms present. In this study, an optimized method for enriching apelin peptides with cationexchange beads followed with mass spectrometry analysis is presented. Apelin peptides are labile in plasma at physiological conditions; however, by lowering the plasma pH to 4.5, the recovery of apelin peptides can be increased significantly. Through optimizing the cation-exchange extraction process, we improved the lower limit of detection for most of the apelin peptides monitored to a few pg/ml. Using the improved method, we detected pyroglutamyl apelin-13 [(pyr)apelin-13] as the major apelin isoform present in plasma from several healthy volunteers at concentrations ranging from 7.7 to 23.3 pg/ml. Ó 2013 Elsevier Inc. All rights reserved.

Apelin, the endogenous ligand for the apelin receptor, is an important hormone regulating cardiovascular homoeostasis [1–3]. Intravenous administration of apelin in rodents and in humans results in reduced blood pressure through vasodilatation [4–6]. Apelin is also a potent inotropic agent for the heart [7]. Intracoronary infusion of apelin stimulates cardiac contractility, resulting in increased coronary blood flow, lower arterial pressure, and peripheral vascular resistance [5]. Apelin has also been studied as an important biomarker for heart failure [5]. Patients with chronic heart failure have lower levels of circulating apelin peptides [8], and infusion of apelin to heart failure patients significantly increases cardiac output, suggesting that apelin or related apelin receptor agonists may serve as therapeutic agents for patients with heart failure [5]. Apelin and its receptor are ubiquitously distributed in many organs, with the highest expression detected in lung, heart, adipose, mammary gland, and brain [9–12]. Besides its important role in cardiovascular homeostasis, apelin is also involved in fluid homoeostasis, glucose metabolism, and other important physiological activities [2]. Apelin was originally isolated from bovine stomach extract and shown to contain 36 amino acid residues (apelin-36). The genes encoding apelin were subsequently cloned from several species to reveal that apelin-36 was derived from a preproprotein containing 77 amino acid residues [13]. The N-terminal 22 residues of the ⇑ Corresponding author. Fax: +1 317 651 1332. E-mail address: [email protected] (E.Y. Zhen). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.07.006

preproprotein contained several hydrophobic amino acid residues, proposed to represent the secretory signal sequence [13]. Apelin36 is located at the C-terminal region of the preproprotein, with the C-terminal 23 amino acid residues being highly conserved in multiple species [11,14]. In apelin-36, approximately one-third of the residues are basic, making it highly positively charged at physiological conditions. The presence of these positively charged residues makes it susceptible to cleavage by serine proteases during posttranslational processing. Indeed, a short form, pyroglutamyl apelin-13 [(pyr)apelin-13],1 containing the C-terminal 13 amino acid residues with the N-terminal glutamine residue cyclized, was identified to be the predominant apelin isoform in human heart together with apelin-13 [15]. In both human and rat plasma, apelin-17, containing the last 17 amino acid residues, and (pyr)apelin-13 were found to be the major forms [16,17]. Apelin was found to be abundantly expressed in bovine and human colostrum and milk [11,18,19]. Mass spectrometry (MS) analyses revealed that apelin in bovine colostrum and milk displayed great structure heterogene-

1 Abbreviations used: (pyr)apelin-13, pyroglutamyl apelin-13; MS, mass spectrometry; CHO, Chinese hamster ovary; EIA, enzyme immunoassay; HPLC, high-performance liquid chromatography; LLOD, lower limit of detection; SIL, stable isotopelabeled; AAA, amino acid analysis; WCX, weak-cation exchange; SCX, strong-cation exchange; MALDI–TOF, a matrix-assisted laser desorption/ionization time-of-flight; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; ACN, acetonitrile; LC–MRM, liquid chromatography–multiple reaction monitoring; RE, relative error; CV, coefficient of variation; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; ACE2, angiotensin-converting enzyme 2.

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ity, with 46 unique forms detected ranging in sizes from 12 to 55 residues due to processing at both amino and carboxyl termini [19]. Both apelin-36 and apelin-13 are capable of promoting extracellular acidification in Chinese hamster ovary (CHO) cells expressing the apelin receptor and lowering blood pressure through intravenous administration [5,13]. Apelin-12 is the shortest form that retains activity [20], suggesting that the C-terminal 12 amino acids are essential for receptor binding and for biological activity. Although both the short and long forms of apelin display similar functions, they differ in tissue distribution, potency, and receptor binding affinities [6,10,18]. In rat, apelin-36 was identified to be the major component in lungs, testis, and uterus, and both the long and short forms are present in the mammary gland [9]. The short apelin peptides were found to be more potent in lowering blood pressure and in the extracellular acidification rate-promoting activity of the CHO cells expressing the apelin receptor [10,13,18]. The fate of internalized apelin receptors is also different when interacting with different apelin forms [21]. With apelin-13, internalized receptors were recycled to the cell surface; in contrast, with apelin-36, the receptors were sequestered intracellularly [21]. With the C-terminal sequence of apelin involved in receptor binding, the N-terminal sequence may modulate its interaction with the receptor. To better understand the biological functions of apelin, it is important to accurately determine the specific forms present and their concentrations. Methods for apelin detection include enzyme immunoassays (EIAs) and radioimmunoassays (RIAs) [16,17]. Most assays use antibodies to target the conserved C-terminal domain in these peptides, effectively measuring the total amount of apelin present. To characterize the specific forms of apelin present, gel filtration or high-performance liquid chromatography (HPLC) separation approaches coupled with apelin-like immunoreactivity detection have been used routinely. Using these approaches, the circulating concentrations for apelin show substantial disparity from a few picograms per milliliter (pg/ml) to several nanograms per milliliter (ng/ml) [8,15,22]. MS approaches, because of their high selectivity, have proved to be effective alternative methods for peptide or protein quantification and characterization [23,24], and they are ideal orthogonal tools for apelin profiling and quantification. Mesmin and coworkers developed an MS-based method for apelin characterization and quantification [19,25]. Taking advantage of the fact that apelin peptides are positively charged under physiological conditions, Mesmin and coworkers first en-

riched apelin peptides in plasma using cation-exchange beads, and the enriched peptides were subsequently characterized or quantified using MS. In one study, the apelin concentrations for the same samples measured using MS and a widely used EIA method were compared. Using EIA, the apelin concentrations in healthy human volunteers were reported to be 200 to 400 pg/ml; however, the MS method failed to detect a single apelin form, although the MS lower limit of detection (LLOD) for the apelin peptides was much lower than the values determined by EIA from these volunteers [25]. These disparate results require additional studies to understand this discrepancy in detail. Building on the original work by Mesmin and coworkers, here we report a modified MS-based method for apelin with improved sensitivity. Using this improved approach, we detected the presence of (pyr)apelin-13 as the major apelin isoform in human plasma.

Materials and methods Chemicals and reagents Both the stable isotope-labeled (SIL) and unlabeled apelin peptides [apelin-36, apelin-17, apelin-13, (pyr)apelin-13, and apelin12] were synthesized by CPC Scientific (Sunnyvale, CA, USA). The following residues were selectively labeled using 13C/15N-labeled amino acids in the corresponding sequences: Leu28 (+7 Da) and Pro35 (+6 Da) in apelin-36, Phe2 (+10 Da) in apelin-17, Leu5 (+7 Da) in apelin-13 and (pyr)apelin-13, and Leu4 (+7 Da) in apelin-12 (Table 1). The purities of the synthesized peptides were more than 95% based on HPLC analysis, and the exact peptide amounts for these standards were determined using amino acid analysis (AAA). The peptide concentrations determined by AAA were used for all subsequent studies. An apelin stock solution containing the five apelin SIL peptides was prepared with each peptide at 200 lg/ml concentration. The stock solution was stored in aliquots at 80 °C. Dynabeads MyOne Carboxylic Acid magnetic beads, weak-cation exchange (WCX) beads, were purchased from Life Technologies (Carlsbad, CA, USA), and strong-cation exchange (SCX) magnetic beads were purchased from Bioclone (San Diego, CA, USA). Polaris 3 C18-A HPLC columns (100  2.0 mm) were obtained from Agilent Technologies (Santa Clara, CA, USA). An apelin12 fluorescent EIA kit (cat. no. FEK-057-23) and apelin-12 purified immunoglobulin G (IgG) antibody (cat. no. G-057-23) were ob-

Table 1 Proteolytic processing of apelin peptides in human plasma after 1 h of incubation at 37 °C.

Note: The amino acid sequences for the intact peptides are listed at top of each section, and the observed degradation products for the corresponding peptide are listed below. The peptide product numbering systems follow the original intact form. The major degradation products based on the MS intensity for each peptide are labeled with an asterisk (⁄) next to their sequence numbers. pE refers to pyroglutamic acid.

Apelin peptide quantification in human plasma / E.Y. Zhen et al. / Anal. Biochem. 442 (2013) 1–9

tained from Phoenix Pharmaceuticals (Burlingame, CA, USA). Pooled human plasma was purchased from Biological Specialty (Reading, PA, USA). Roche Complete protease inhibitor was purchased from Roche Diagnostics (Indianapolis, IN, USA). The inhibitor was used directly according to the manufacturer’s recommendations without further testing. Formic acid (98%) was obtained from Sigma–Aldrich (St. Louis, MO, USA). Apelin degradation in human plasma To 200 ll of human plasma, 2 lg of the five unlabeled apelin peptides was spiked in separately. The samples were incubated at 37 °C for 1 h, and the apelin degradation products were enriched using WCX beads as described below. The samples were analyzed using a matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) instrument (see below). Human plasma collection Healthy human plasma was collected from consented donors with approval from the Lilly Multidisciplinary Program Oversight Committee. For six healthy donors (three male and three female, 37–71 years of age) with no known underlying health conditions, 10 ml of blood was drawn into a K2EDTA tube (BD Diagnostics, Franklin, NJ, USA) from each donor. Immediately after blood collection, apelin SIL peptide standards in formic acid solution were spiked into the blood collection tubes using syringes. The blood collection tubes were mixed thoroughly and placed on ice. To prepare the apelin SIL peptide standards in formic acid, 40 ll of the MS plasma matrix (see below) was mixed with an equal volume of formic acid (98%) first, and apelin SIL peptides at 1 ng each for the five peptides were added to the solution. The presence of human plasma in the SIL/formic acid solution was used to reduce the loss of the apelin peptides due to nonspecific binding. The final concentration of each SIL peptide in the 10 ml of blood collected was 100 pg/ ml. Within 30 min of blood collection, the blood tubes were centrifuged at 3000g for 10 min to collect plasma. The resulting plasma pH was approximately 5.0–5.5. Plasma was analyzed shortly after without being subjected to freeze/thaw cycles. Apelin enrichment Cation-exchange magnetic beads were used for apelin enrichment from plasma. The WCX beads were used directly out of the vial. The SCX beads were resuspended in 20% ethanol to a concentration of 20 mg/ml. The beads were washed twice using a phosphate-buffered saline (PBS) solution and once in assay buffer (10 mM Tris and 10 mM Hepes, pH 7.4). To 500 ll of the acidified human plasma, 100 ll of beads was added, and the samples were diluted to a final volume of 1 ml using 4 °C assay buffer. The samples were allowed to mix gently at 4 °C for 15 min, and the supernatants were removed with the aid of a magnet. The beads were washed three times using 1 ml of the cold assay buffer, and the enriched peptides were eluted using 60 ll of elution solution composed of 1% trifluoroacetic acid (TFA), 5% acetonitrile (ACN), and 1 M KCl. The supernatants containing the apelin peptides were transferred to HPLC vials for MS analyses.

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to sit in ice for 15 min, and precipitated protein was removed through centrifugation at 15,000g. The acidified plasma was used for all subsequent spike-in and recovery experiments. Apelin SIL peptides were added to the acidified MS plasma matrix at 100 pg/ml for each peptide, and this solution was used for serial dilutions of unlabeled apelin peptides. Two sets of samples with 2-fold serial dilutions of the unlabeled peptides were prepared, with the nine calibration samples ranging from 0.98 to 250 pg/ml and eight validation samples ranging from 1.56 to 200 pg/ml. Each validation data point was bracketed by two calibration data points. The samples with 100 pg/ml SIL peptides but without any spikedin unlabeled peptides were used as blank controls. A volume of 500 ll for each sample was analyzed in duplicate. MS analysis To measure apelin peptide forms by MS, samples were analyzed with an AB SCIEX Qtrap 5500 MS (AB Sciex, Framingham, MA, USA) using a liquid chromatography–multiple reaction monitoring (LC– MRM) method specific for the targeted apelin peptides. Samples were loaded onto a Polaris 3 C18-A HPLC column (100  2.0 mm) using a Shimadzu SIL-30AC autosampler. The column was kept at 50 °C in a CTO-30A oven, and the gradient was generated using an LC-30AD HPLC pump at a flow rate of 250 ll/min. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in ACN. The HPLC gradient was as follows: 0–4 min, 5% B; 4.1– 10 min, 10–25% B; 10–10.2 min, 25–70% B; 10.2–11 min, 70% B; 11–11.2 min, 70–5% B; 11.2–15 min, 5% B. The MS settings were as follows: CUR, 20; CAD, medium; IS, 5500 V; TEM, 500 °C; GS1, 60; GS2, 60. The settings for individual transitions are listed in Table S1 of the supplementary material, with data collected 60 s around the corresponding retention time. For MALDI–TOF MS analyses, an AB SCIEX TOF/TOF (tandem time-of-flight) 5800 instrument was used with a-cyano-4hydroxycinnamic acid (Sigma–Aldrich) as the matrix. All samples were analyzed in reflector mode using laser settings that do not induce in-source fragmentation. Apelin form profile in human plasma To two donor samples, (pyr)apelin-13 SIL peptide in formic acid was added to the blood immediately after drawing to a concentration of 500 pg/ml. Then 3 ml of the plasma was used for apelin enrichment with 200 ll of WCX beads. The eluted material from WCX beads was desalted using a lC18 ZipTip (Millipore, Billerica, MA, USA), and one-third of the sample was loaded onto a PicoFrit capillary column (75 lm i.d.  7 cm) with a 15-lm spray tip packed with YMC ODS C18 resin and analyzed using a Thermo LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) following the procedure described by Higgs and coworkers [26]. Peptide identification from the data-dependent LC–MS experiments was conducted using an in-house program that uses a statistical wrapper to postprocess the output of the OMSSA, X! Tandem, and Protein Pilot software programs with a decoy database strategy of reversed protein sequences to filter out false positive identifications [26].

Preparation of calibration and validation samples

Results

For MS spike-in and recovery experiments, the purchased pooled human plasma was used as the matrix, and the endogenous apelin peptides were depleted by incubating the plasma at 37 °C overnight followed by acidification to pH 4.5 using formic acid (MS plasma matrix). To achieve the desired pH, 2 ll of formic acid (98%) was added to every milliliter of plasma. Plasma was allowed

Apelin degradation study Human plasma is rich in a variety of different kinds of proteases, and peptides can be degraded quickly. To accurately quantify endogenous apelin peptides, it is necessary to understand the degradation of these peptides to devise a method to stabilize them

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during analysis. To study apelin degradation in human plasma, apelin peptides were spiked into human plasma, and the samples were incubated at 37 °C for 1 h. Proteolytically processed apelin peptides were enriched and analyzed using MALDI–TOF MS. Significant peptide processing was detected for all peptides tested. The processed products detected for each peptide are listed in Table 1. For most peptides, except (pyr)apelin-13, significant degradation was detected at their respective N termini, resulting in systematically processed forms ending at the last arginine residue of the apelin peptide sequence. This processing pattern is similar to those produced by plasma aminopeptidases. For apelin-36, the most abundant products (1–22, 6–36, 9–36, and 23–36) all resulted from cleavage after an arginine residue, likely attributed to serine proteases. Carboxypeptidase activity was less pronounced, with the main product observed for all five apelin peptides consisting of cleavage after the fourth proline residue from the C terminus (Table 1). A similar C-terminal cleavage product was also detected in the bovine colostrum study [19]. For (pyr)apelin-13, because the N terminus was protected by the cyclization of the glutamine residue, the only degradation detected was at the C terminus. Both apelin-36 and apelin-17 can be processed to apelin13 and apelin-12, which were observed in our studies. Apelin stability study As shown above, apelin in plasma can be degraded quickly by endogenous plasma proteases. To prevent artifactual proteolytic processing of apelin, different methods were evaluated by using a broad spectrum of protease inhibitors, by acidification of the plasma to inactivate proteolytic enzymes, or by combining both approaches. Human plasma at its normal pH (7.4) or acidified to pH 4.5 with formic acid was spiked with an apelin peptide mixture consisting of apelin-36, apelin-17, apelin-13, and (pyr)apelin-13 at 1 ng/ml concentration. A protease inhibitor cocktail (Roche) was added to selected samples at twice the recommended concentration. The samples were incubated at room temperature for 0, 15, 30, 60, and 120 min. A 250-ll aliquot of each sample was taken at different time points for apelin analyses using WCX beads. A fixed amount of apelin SIL peptide was added to the samples just before MS analysis. As shown in Fig. 1, at physiological pH, all four spiked-in peptides were degraded quickly, with more than 50% of the apelin peptides disappearing within 30 min. The addition of protease inhibitors slowed the degradation slightly, and within 2 h all of the intact forms were gone. As expected, (pyr)apelin-13, with the protection of the pyroglutamic acid residue at the N-terminal end, was most stable among the four peptides, and apelin-36 was the most labile peptide owing to its longer sequence with more protease cleavage sites. Apelin-12 was not spiked into the samples; however, the formation of the apelin-12 was monitored from the degradation of longer peptides. As shown in Fig. 1, apelin-12 was quickly formed and reached its maximum concentration after 15 min, after which it was further degraded under those conditions at physiological pH. In contrast, at pH 4.5, the four spiked-in peptides were readily detected throughout the entire study period, with more than 85% of the total apelin signals recovered (Fig. 1). There was minimal apelin-12 formation in the acidified samples (Fig. 1). The presence of protease inhibitors improved the stability slightly for the four peptides under acidic pH; however, acidifying the plasma to pH 4.5 was determined to be an easy and effective method to stabilize apelin peptides in plasma. Method optimization for apelin enrichment Apelin peptides are rich in basic residues, and in apelin-36 alone nearly one-third of the residues plus the N-terminal amino

group are positively charged at neutral pH. (Pyr)apelin-13, with the fewest number of basic residues among the five peptides, still has 4 net positive charges, which makes apelin peptides ideal targets for enrichment using cation-exchange approaches. Two methods for apelin enrichment were compared initially for the recovery of the spiked-in apelin peptides. The first used anti-apelin-12 antibody coupled beads, and the second used WCX beads as demonstrated by Mesmin and coworkers [25]. The cation-exchange extraction method, using either WCX or SCX beads, proved to be much more efficient than the antibody-based approach, resulting in better sensitivity (data not shown). In addition, the short incubation time associated with cation exchange minimizes apelin peptide degradation in plasma during sample incubation. Unfortunately, Mesmin and coworkers were not able to detect apelin peptides in human plasma using the WCX method [25]. To extend the utility of this approach, we embarked on a rigorous evaluation of ion exchange methods. Apelin peptide recovery was first evaluated using either WCX or SCX beads under different elution conditions. In these experiments, plasma with spiked apelin peptides was diluted 2-fold using PBS before being subjected to cation-exchange enrichment with PBS as the washing buffer. For apelin-36, using either WCX or SCX beads, the acidic elution condition (5% ACN/1% TFA/1 M KCl, pH 1.0) was found to be the best, with a 40-fold higher recovery than the neutral elution condition (PBS/5% ACN/1 M KCl, pH 7.4), whereas WCX beads allowed for an approximately 3-fold higher recovery than SCX beads (data not shown). For apelin-17, the yield was also higher when using WCX beads. In contrast, shorter peptides, with fewer basic residues, tend to have higher recovery yields with SCX beads, and any elution condition tested provided effective recovery. (Pyr)apelin-13 had the lowest recovery using WCX beads, observed to be approximately 40% (Fig. 2). The biggest improvement on short peptide recovery was attributed to bead washing conditions. Using WCX beads, when the plasma solution was diluted first and the beads were washed using the low ionic strength assay buffer, the yield for (pyr)apelin-13 was improved approximately 2-fold. The plasma pH effect on peptide recovery was also evaluated by adjusting the plasma pH to 2.7, 3.5, 4.5, and 7.4. At pH 2.7, recoveries for all peptides using WCX beads were quite low, and for the three short peptides the observed recoveries were less than 10%, whereas the yields at pH 4.5 and 7.4 for most peptides were comparable, with (pyr)apelin13 having the best recovery at pH 4.5 (Fig. 2). To balance the peptide recovery for both long and short forms, WCX beads were used in all subsequent studies with the MS plasma matrix acidified to pH 4.5 and whole blood from healthy donor samples adjusted to a pH of 5.0–5.5 (see Discussion). The acidified plasma was diluted with an equal volume of the assay buffer, and the final pH during WCX bead extraction was approximately 5.5–6.0. The peptides were eluted using the acidic elution buffer. Under these conditions, 500 ll of plasma with 200 pg/ml peptides spiked in yielded recoveries for apelin-12, apelin-13, (pyr)apelin-13, apelin-17, and apelin-36 of 90%, 82%, 78%, 83%, and 94%, respectively (Fig. 2). Method validation To validate the overall apelin enrichment and detection method, an MS plasma matrix was prepared by incubating the pooled human plasma overnight at 37 °C to proteolyze endogenous apelin peptides. LC–MRM analysis showed that in this MS plasma matrix, the endogenous levels of the five apelin peptides monitored were undetectable. Following the criteria recommended for bioanalytical method validation [27], four interday runs with two replicates within each run were performed. Two types of samples, calibration and validation, were included in the studies, as shown in Fig. 3. The

Apelin peptide quantification in human plasma / E.Y. Zhen et al. / Anal. Biochem. 442 (2013) 1–9

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Fig.1. Apelin peptide processing in human plasma under acidified and normal conditions. Apelin-13, (pyr)apelin-13, apelin-17, and apelin-36 at 1 ng/ml were spiked into human plasma at pH 4.5 or 7.4 with and without the Roche protease inhibitor. The samples were incubated at room temperature for varying periods of time. Apelin peptides were enriched using WCX beads, and a fixed amount of SIL peptides was spiked into the samples before LC–MRM analysis. Degradation of the four spiked-in peptides and the formation of apelin-12 were monitored. The ratios between the unlabeled and SIL peptides are plotted.

peptide concentrations in the calibration samples ranged from 0.98 to 250 pg/ml, and those in the validation samples ranged from 1.56 to 200 pg/ml. A fixed amount of SIL peptides (100 pg/ml) for each peptide was present in all samples. Concentration–response curves were generated from calibration samples using the area ratios between the unlabeled and SIL peptides and fit to a four-parameter logistic function on the log concentration scale. The concentrations for validation samples were back-calculated from the curve fits. The bias (accuracy) and variability (precision) of the assay were quantified as relative error (%RE) and coefficient of variation (%CV), respectively. Total error is the sum of the absolute value of %RE and %CV. The plots for %RE, %CV, and the total error of the five apelin peptides are shown in Fig. 4. The following common acceptance criteria were used to determine the lower limit of quantification (LLOQ):%RE and %CV within ±20% [+25% at LLOQ/ ULOQ (upper limit of quantification)] and total error within ±30% [27]. Following these criteria, the LLOQ, ULOQ, and LLOD for each peptide were determined as shown in Fig. 4. For the three short apelin peptides apelin-12, apelin-13, and (pyr)apelin-13, the LLOQs were 3.18, 1.59, and 6.25 pg/ml, respectively. The LLOQs for apelin17 and apelin-36 were 25 and 100 pg/ml, respectively (Fig. 4). The ULOQs for most peptides, except apelin-17, were 200 pg/ml. The ULOQ for apelin-17 was 100 pg/ml (Fig. 4). The two longer peptides, especially apelin-36, did not ionize as well as the shorter peptides. Furthermore, the larger peptides exhibited greater loss from nonspecific binding. Together, these factors contributed to the relatively high LLOQ values for apelin-17 and apelin-36. The LLOD for most of the peptides was roughly 1 pg/ml (Fig. 4).

Apelin in healthy volunteers Apelin from six healthy volunteers was measured using both the Phoenix EIA kit and the LC–MRM method. Using the EIA kit, the measured values ranged from 49.3 to 273 pg/ml (Table 2). Because the antibody in the EIA kit is reported to cross-react with each of the apelin forms analyzed in this study, the kit effectively measures total apelin concentration. The information provided with the kit did not mention whether the antibody cross-reacts with any apelin forms shorter than apelin-12. However, using LC–MRM, only (pyr)apelin-13 forms were positively identified in five of the six samples in the range of 7.7–23.3 pg/ml (Table 2). The difference on (pyr)apelin-13 concentration detected in this study reflects individual variation. In one of the human samples, (pyr)apelin-13 was not detected (Table 2). The endogenous (pyr)apelin-13 had the same retention time and a similar MRM transition profile as the SIL peptide. The extracted ion chromatograms for (pyr)apelin-13 from one donor are shown in Fig. 5. The MS-measured apelin concentrations were 4- to 20-fold lower than the values measured using EIA, and there is no direct correlation between the two sets of values (Table 2). Trace amounts of apelin-12 close to the LLOD level were also identified in all six samples; however, the signal for apelin-12 was quite weak (data not shown). Apelin-36, apelin-17, and apelin-13 were not detected in any of the samples (data not shown). The recovery for the apelin SIL peptides spiked into whole blood at 100 pg/ml was also evaluated by comparing the SIL peptide signals measured from the six donor samples with those measured

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Fig.2. Apelin peptide enrichment optimization using cation-exchange beads. The effects of different sample dilution buffers (A), cation-exchange beads (WCX vs. SCX) (B), and sample pH (C) on peptide enrichment were evaluated. To human plasma with endogenous apelin peptides depleted, the pH of the samples was adjusted to different values using formic acid. Unlabeled apelin peptide standards were spiked in at 200 pg/ml, and 0.5 ml of the samples was diluted to 1 ml using PBS or 10 mM Tris and 10 mM Hepes (pH 7.4). Peptides were enriched using either WCX or WCX beads. After elution, 250 pg of labeled apelin peptides was spiked into elutes before LC–MRM analysis, and the recovery yields for each peptide under different conditions were calculated.

from the 18 calibration samples used in the same study, also at 100 pg/ml. The average recovery values for apelin-12, apelin-13, (pyr)apelin-13, apelin-17, and apelin-36 were 68%, 51%, 75%, 60%, and 25%, respectively (see Table S2 in supplementary material). Among the five peptides, only (pyr)apelin-13 maintained a similar yield recovering either from acidified whole blood or plasma. The recovery for apelin-36 is significantly lower in acidified whole blood versus plasma. The recovery difference observed here for each peptide emphasizes the importance of adding the control peptides immediately after blood collection. The LC–MRM method focused on five apelin peptides for identification and quantification. To assess whether other apelin forms were present in human circulation, apelin peptides in 3-ml samples were enriched from two donors, with (pyr)apelin-13 SIL spiked in as internal control. The enriched material was analyzed using the data-dependent LC–MS method on an LTQ-Orbitrap Velos mass spectrometer. In this study, there was no positive identification for any apelin forms. This was not totally unexpected given that the apelin peptides tend to display as +4 to +9 charged ions, and none of the database searching programs used in this study can handle ions with charge states larger than +4. In the bovine colostrum study, 46 unique bovine apelin forms were identified, with their m/z values reported previously [19]. For bovine and human apelin peptides, the C-terminal 27 residues are conserved in both species, and among the 46 bovine apelin peptides, 24 peptides are located in the conserved region. Manual data analyses for the 24 peptides previously reported were performed for these two donor samples. However, no apelin peptides were identified with a mass tolerance of 10 ppm, whereas the spiked-in (pyr)apelin-13 SIL peptide was positively identified. No endogenous (pyr)apelin13 was identified in these two samples, suggesting that the Orbitrap Velos-based approach has lower sensitivity than the LC–MRM method.

Discussion Apelin and its receptor play important physiological roles in the cardiovascular systems, affecting cardiac contractility and blood pressure. Apelin peptides also affect the pancreatic islets function influencing insulin secretion [28] and body fluid homeostasis [3], so accurate determination of apelin isoforms and concentration in circulation is critical to understand its diverse functions. A sensitive MS-based method to study apelin concentration in circulation has been developed with the apelin peptides first enriched using cation-exchange beads [25]. In this study, we further optimized the method in three aspects: (i) improved stabilization of endogenous apelin peptides through acidifying human plasma, (ii) added control peptides immediately after blood collection, and (iii) further optimized the cation-exchange enrichment process for the distinct apelin peptide forms. Apelin peptides are labile in human plasma. In circulation, these peptides are reported to be rapidly cleared within a few minutes in vivo [6]. In our current in vitro studies with a relatively high level of spiked-in apelin peptide standards, more than 50% of the spiked peptides are lost in less than 30 min (Fig. 1). Effective stabilization of the endogenous peptides is critical for accurate determination of their concentrations. In our degradation study, apelin peptides were quickly processed by the combined activities of endogenous circulating proteases. Using a combination of several inhibitors targeting the general spectrum of known proteases, including serine, cysteine, and aspartic proteases and metalloproteases, Mesmin and coworkers showed that the stability for most of the apelin peptides can be improved significantly [25]. Unfortunately, we detected only a marginal increase in the stability of the apelin peptides in the presence of Roche protease inhibitors that specifically inhibit many of these proteases. A simple and more effective method for stabilizing apelin peptides was achieved by

Apelin peptide quantification in human plasma / E.Y. Zhen et al. / Anal. Biochem. 442 (2013) 1–9

Fig.3. Apelin method validation using LC–MRM coupled with WCX enrichment. Representative results for (pyr)apelin-13 are shown. Two sets of samples for calibration (solid square) and validation (open circles) were prepared and analyzed using the methods developed. The (pyr)apelin-13 concentrations ranged from 0.98 to 250 pg/ml in the calibration samples and from 1.56 to 200 pg/ml in the validation samples (A). In all samples, the SIL peptide concentration was 100 pg/ml (B). IS, internal standard. The area ratios between unlabeled and SIL apelin peptides were calculated (C), and concentration–response curves were generated from the ratios to back-calculate the measured peptide concentrations in the validation samples. Duplicate samples were analyzed at each concentration.

plasma acidification. Most proteases, except aspartic proteases, have an optimal pH of neutral or basic, so lowering the pH of the plasma effectively inhibits their activities. A similar approach has been used to successfully stabilize acylated ghrelin in plasma [24]. In the current study, we found that lowering the pH of plasma to pH 4.5 significantly improves the detection and recovery of several apelin peptides. With formic acid added immediately after blood collection, it is expected that most protease activities were effectively inhibited. Together with formic acid, apelin SIL peptides were also added to serve as controls to account for potential deg-

7

radation during sample processing. In the original method employed by Mesmin and coworkers, the inhibitors were added after the centrifugation step, and we speculate that significant peptide degradation occurs between sample collection and centrifugation steps. In our studies, samples were subjected to peptide enrichment within 1 h after collection and stabilization, as opposed to being frozen first and analyzed later [25]. Hemolysis was observed due to the addition of formic acid; however, this did not interfere with apelin peptide recovery, as assessed in our quantitative studies. The amount of formic acid added to the whole blood affects the final plasma pH, but it also affects the amount of plasma released after centrifugation. High levels of formic acid in blood result in complete protein aggregation with low plasma released after centrifugation. To achieve the right balance of sample acidification and sufficient plasma released, a ratio of 4 ll of formic acid (98%) per milliliter of whole blood was used, which results in a final pH of 5.0–5.5. Under these conditions, 3–4 ml of plasma from 10 ml of blood is readily collected. Doubling the amount of formic acid to 8 ll per milliliter of blood resulted in no plasma released after centrifugation. For human plasma, 2 ll of formic acid per milliliter of plasma achieves a pH level of 4.5. The condition of acidifying plasma to pH 4.5 does not interfere with the WCX binding. At these pH levels, yields for the apelin-12, apelin-17, and (pyr)apelin-13 peptides were higher than those obtained at neutral pH conditions (Fig. 2). Apelin is synthesized as a preproprotein of 77 amino acids, and sequence analysis suggests that it is processed into a proprotein of 55 amino acids, which was recently identified in bovine colostrum by MS [19]. The full-length preproprotein has also been detected as a dimer in cardiac tissues of wild-type and preproapelin transgenic mice [4]. The preproprotein dimer was stabilized through a disulfide bond. Lacking cysteine residues, the short peptides are most likely present as monomers [4]. The majority of apelin peptides identified in plasma and in different tissues are the short forms; however, limited information is available for the processing of the proapelin protein and the catabolism of apelin peptides. The only known enzyme that can cleave apelin is angiotensin-converting enzyme 2 (ACE2), which hydrolyzes the C-terminal phenylalanine residue from apelin-13 and apelin-36 peptides [29]. In bovine colostrum and milk, many forms of apelin were detected with the C-terminal phenylalanine residue removed, likely the result of ACE2 activity [19]. Deletion of the last phenylalanine residue [30,31] or its replacement with alanine [4] leads to the loss of the hypotensive activity and its ability to induce apelin receptor internalization [31]. In our short degradation study in plasma, we did not detect any apelin products from ACE2 activity. This may be due to the short duration of our studies and potentially low ACE2 activity in plasma samples. Cation-exchange extraction is based on charge–charge interaction, and the buffer ionic strength influences the interaction. In the original method described by Mesmin and coworkers, PBS solution was used to wash the WCX beads after sample incubation [25]. In our study, washing these WCX beads with PBS reduces the yields significantly, especially for (pyr)apelin-13, because it has the weakest binding strength among the five apelin peptides. With the substitution of a low-ionic strength buffer for PBS, the yields for apelin-12, apelin-13, (pyr)apelin-13, apelin-17, and apelin-36 in plasma were 90%, 82%, 78%, 83%, and 94%, respectively, as compared with 71%, 24%, 16%, 68%, and 71%, respectively, as reported by Mesmin and coworkers [25], resulting in a 5-fold increase for (pyr)apelin-13. The overall LLOQ for the optimized method described in this study is much lower than that reported by Mesmin and coworkers [25]. This optimized protocol enabled us to detect (pyr)apelin-13 as the major form in circulation. The presence of (pyr)apelin-13 is likely due to its longer half-life as to other forms

8

Apelin peptide quantification in human plasma / E.Y. Zhen et al. / Anal. Biochem. 442 (2013) 1–9

Fig.4. Bias (%RE), precision (%CV), and total error (%RE +%CV) estimated from four sets of interday spiked and recovery experiments for the five apelin peptides monitored. The following common acceptance criteria were used to determine LLOQ and ULOQ: %RE and %CV within ±20% (+25% at LLOQ/ULOQ) and total error within ± 30%. The LLOQ, ULOQ, and LLOD values for each peptide are listed on top of each plot.

Table 2 Apelin measured using EIA and MS from healthy volunteers. Sample

EIA (pg/ml): Total

MS (pg/ml): (Pyr)apelin-13

1 2 3 4 5 6

102.9 ± 23.6 49.3 ± 4.7 59.0 ± 13.0 98.9 ± 12.8 192.1 ± 47.0 273.1 ± 21.1


Fig.5. Representative plots for the extracted ion chromatograms for (pyr)apelin-13 from one healthy volunteer. The upper panel is the spectrum for the endogenous apelin peptide, and the lower panel is the spectrum for the corresponding spiked-in SIL peptide.

[32]. (Pyr)apelin-13 has been identified in several studies to be the major form present in human and rat plasma and in rat brain [16,17], which is consistent with the results from our studies. Among the five apelin peptides analyzed, apelin-36 is the longest one. The greater amino acid sequence length for apelin-36 makes its ionization process less efficient as compared with the shorter apelin forms. Its positively charged nature makes apelin-36 susceptible to increased losses due to nonspecific binding. These factors, together with its higher propensity for proteolytic degradation, leads to a lower recovery from whole blood and the highest overall LLOQ value for the apelin peptides analyzed in these studies (Fig. 4). The source of the apelin in plasma has not been determined. Apelin was found to be expressed by adipose tissue and in heart [22,33]. In obese people, the apelin concentrations in plasma are elevated [33], and the plasma apelin concentrations in normal individuals and heart failure patients showed significant correlation with the apelin levels in heart atrium [22]. Thus, both adipose tissue and heart can be sources of plasma apelin. The exact form of apelin in adipocytes has not been identified, and the major form of apelin in heart has been reported to be (pyr)apelin-13 [15], which is likely an important source for (pyr)apelin-13 in plasma. Because the plasma concentration of apelin is quite low, it has been suggested that apelin is not a circulating hormone and that it functions in a paracrine fashion [9,34]. Indeed, apelin is highly expressed in several tissues, with significantly higher concentrations than that in plasma [9,17,22]. In bovine stomach, the tissue in which apelin was originally discovered, apelin was found to be present as apelin-36 [13]. HPLC separation of the stomach extract revealed many fractions containing active peaks, suggesting that apelin in bovine stomach is present in more than one form. In human heart, an immunohistochemical study showed that apelin is specifically localized to the endothelium of the vasculature and present as (pyr)apelin-13 with its methionine residue oxidized [15]. To characterize whether the apelin peptides in circulation are also present in the Met-oxidized forms, we developed an LC– MRM method targeting all five apelin peptides in the Met-oxidized forms and analyzed several samples from healthy volunteers; however, we failed to detect the presence of any Met-oxidized form (data not shown). The apelin concentrations measured using EIA are much higher than the values determined by MS, corroborating the observation reported by Mesmin and coworkers [25]. EIA measures apelin

Apelin peptide quantification in human plasma / E.Y. Zhen et al. / Anal. Biochem. 442 (2013) 1–9

immunoreactivity, and the antibody used in the assay is known to react with all five apelin forms monitored in this study; it may also react with other forms of apelin, including forms less than 12 residues in length, not readily detected by our MS methods. Our method reliably detects the known biologically active forms of apelin. In summary, we have found that apelin peptides can be proteolytically processed quickly in plasma and that lowering the pH of the plasma confers significant stability to endogenous apelin peptides. With the improved cation-exchange extraction method, we successfully identified that (pyr)apelin-13 was the major form of apelin present in human plasma and that a trace amount of apelin-12 was also present. The positive identification of the presence of (pyr)apelin-13 as the major form in plasma will help to understand the biological function of this important hormone.

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Acknowledgment

[19]

We thank Thomas P. Beyer, Patricia Foxworthy, and Laura F. Michael for their advice and collaboration.

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