Selective electromembrane extraction based on isoelectric point: Fundamental studies with angiotensin II antipeptide as model analyte

Journal of Membrane Science 481 (2015) 115–123

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Selective electromembrane extraction based on isoelectric point: Fundamental studies with angiotensin II antipeptide as model analyte Chuixiu Huang a,b, Astrid Gjelstad a, Stig Pedersen-Bjergaard a,c,n a

School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway G&T Septech AS, PO Box 33, 1917 Ytre Enebakk, Norway c Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark b

art ic l e i nf o

a b s t r a c t

Article history: Received 7 January 2015 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online 17 February 2015

For the first time, selective isolation of a target peptide based on the isoelectric point (pI) was achieved using a two-step electromembrane extraction (EME) approach with a thin flat membrane-based EME device. In this approach, step 1 was an extraction process, where both the target peptide angiotensin II antipeptide (AT2 AP, pI ¼5.13) and the matrix peptides (pI 45.13) angiotensin II (AT2), neurotensin (NT), angiotensin I (AT1) and leu-enkephalin (L-Enke) were all extracted as net positive species from the sample (pH 3.50), through a supported liquid membrane (SLM) of 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% (v/v) di-(2-ethylhexyl)-phosphate (DEHP), and into an aqueous acceptor solution (pH 1.80). In step 1, the cathode was located in the acceptor solution. Following step 1 (and prior to step 2), the pH of the acceptor solution was adjusted to pH 5.25, and the anode was located in the acceptor solution. Step 2 was a clean-up process to remove the matrix peptides with pI 45.13 (net positively charged) from the acceptor solution pH 5.25. During step 2, the target peptide was not net positively charged. This suppressed complex formation with negatively charged DEHP, and the target remained in the acceptor solution. The acceptor solution pH, the SLM composition, the extraction voltage, and the extraction time during the clean-up process (step 2) were important factors influencing the separation performance. An acceptor solution pH of 5.25 for the clean-up process slightly above the pI value (pH 5.13) was found to be optimal. Under the optimal conditions, 73% of AT2 AP (RSD 13%) and 48% of L-Enke (RSD 5%) were found in the solution after this two-step EME process, whereas the other three positively charged peptides were not detected. The observations above indicated that two-step EME may have a future potential for the fractionation of peptides and other ampholytic compounds based on their isoelectric points. & 2015 Elsevier B.V. All rights reserved.

Keywords: Supported liquid membrane (SLM) Electromembrane extraction (EME) Peptides Isoelectric point Selective extraction

1. Introduction Electromembrane extraction (EME) was introduced as a microextraction technique in 2006 [1]. In EME, charged analyte molecules in the sample migrate into an acceptor solution through a supported liquid membrane (SLM), where the transport is facilitated by an electric field [2]. Due to the fast electrokinetic migration mechanism, EME is a rapid and efficient sample preparation technique [3,4]. EME can achieve the isolation of analytes, sample clean-up, and potential enrichment in a single step [5]. In addition, the EME device is inexpensive and easy to fabricate n Corresponding author at: School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway. E-mail address: [email protected] (S. Pedersen-Bjergaard).

http://dx.doi.org/10.1016/j.memsci.2015.02.006 0376-7388/& 2015 Elsevier B.V. All rights reserved.

in-house, and EME is simple in operation and friendly to the environment because of very little consumption of organic solvent for each extraction [6]. Because of the advantages listed above, EME has been employed for the extraction of inorganic anions [7], metal ions [8,9], organic pollutants [10,11], acidic drugs [12], acidic herbicides [13], basic drugs [1,14], amino acids [15,16], and small peptides [17,18]. However, EME also faces some challenges, especially for the extraction of amino acids and peptides, such as relatively low EME recovery (10% to 50%) and bubble formation due to electrolysis during the EME process [6,19]. EME of amino acids was first reported in 2011 [15], where 17 amino acids were extracted under constant voltage conditions with an SLM of 1-ethyl-2-nitrobenzene containing 15% bis-(2ethylhexyl) phosphoric acid (v/v).The recoveries for the amino acids with 2.5 M acetic acid in the sample were in the range of 0.5–10.8% (RSD r 13%). The stability and repeatability for EME of

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amino acids was subsequently improved by using constant system-current conditions [20]. After the introduction of pulsed EME (PEME) [21], three amino acids, namely histidine, phenylalanine, and tryptophan were extracted with an SLM of 2-nitrophenyl octyl ether containing 5% di-(2-ethylhexyl) phosphate (DEHP) and 10% tris-(2-ethylhexyl) phosphate (TEHP) (v/v/v) using one-way PEME, and the extraction recoveries were in the range of 7.1–21.6% (RSD r4.5%) [16]. Up to date, a few research articles have reported EME of peptides with the hollow fiber EME format [17,18,22–26]. However, high system-current and low recoveries for the peptides have been the main challenges [6,19]. Most recently, a flat membrane-based EME device was developed, and exhaustive extraction of some basic drugs was achieved [27]. With this new EME format, exhaustive EME of peptides has also been obtained with system-current less than 50 mA [28]. In the latter method, EME was performed for 25 min from 600 mL of phosphate buffer solution (pH 3.5), into 600 mL of 50 mM phosphoric acid as acceptor solution, with an SLM of 1-nonanol diluted 1 to 1 (v/v) with 2-decanone containing 15% DEHP, and with a voltage of 15 V. The recoveries for six model peptides ranged from 77 to 94% (RSDo10%). Differences in isoelectric point (pI) have been widely used for the separation of ampholytic compounds such as amino acids, peptides, and proteins [29–34]. In one example, the isolation of amino acids by iso-electric focusing (IEF) using an electromembrane type process (EMP) was reported [30]. At pH 8 (above the pI value of glutamic acid (GLU), iso-electric separation of GLU from GLU–lysine (LYS) mixture was achieved with a separation factor of approximately 9 (the recovery ratio between GLU and LYS), and the absolute recovery for GLU was 85%. The separation of GLU from GLU–LYS mixtures was mainly due to the differences in pI values resulting in opposite charges. In another example, fractionation of peptides in the wheat gluten hydrolysates by IEF was presented [31]. At pHo7, the content of acidic and neutral peptides in each fraction was over 90%, while when the pH was increased from 8 to 10, the content of acidic and neutral peptides in the fraction was decreased from approximately 90% to 60% as expected. The separation of proteins by IEF has also been reported, where controlled separation of proteins in an aqueous sample solution into acidic and basic fractions using single-component ampholyte buffers with well-defined pI cut-off values was obtained [32]. Most recently, selective EME of tryptophan from a mixture of three amino acids was reported using two-way PEME and based on differences in the isoelectric points [16]. In this two-step approach, the first step involved extraction of all three amino acids as net positive charged species from the sample and into the acceptor solution. Prior to the second step, the pH of the acceptor solution was increased to the pI value of tryptophan (5.89), and the polarity of the electrodes was reversed. During the second step, the two amino acids with pI 4 5.89 were removed from the acceptor solution based on the electrical field, while tryptophan with no net charge was unaffected by the electrical field and principally remained in the acceptor solution [16]. High and predictable selectivity for peptides based on established physico-chemical parameters is mandatory for future acceptance and use of EME. Inspired by selective EME of amino acids as reported recently [16], selective EME of peptides based on differences in the isoelectric point was investigated for the first time in this fundamental work. Angiotensin II antipeptide (AT2 AP) was selected as model analyte and this was separated from four matrix peptides based on differences in isoelectric point using a two-step EME approach. The present work, using buffer solutions as sample, investigated the general work flow principles, and investigated the impact of major operational parameters like acceptor solution pH, SLM composition, voltage and time on the selective extraction of AT2 AP.

2. Experimental 2.1. Chemicals and materials Angiotensin II antipeptide (AT2 AP), angiotensin II acetate (AT2), neurotensin (NT), angiotensin I trifluoroacetate (AT1), and Leuenkephalin (L-Enke) were all purchased from Bachem (Bubendorf, Switzerland). Decanol mixture of isomers (Z98%), n-decyl alcohol (Z98%), 1-nonanol (98%), 1-undecanol (99%), 1-dodecanol (Z99.5%), 2-octyl-dodecanol (97%), 2-nonanone (Z99%), 2-decanone (98%), 2undecanone (99%), 2-nitrophenyl octyl ether (Z99%), 2,4-dimethyl-1nitrobenzene (Z99%), 1-ethyl-2-nitrobenzene (Z99%), and di-(2ethylhexyl)-phosphate (DEHP) (Z95%) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was purified with a Milli-Q water purification system (Molsheim, France). Formic acid (98–100%), sodium formate (Pro analysis), acetic acid (ACS), sodium acetate (Pro analysis), phosphoric acid (Pro analysis), sodium dihydrogen phosphate monohydrate (Pro analysis), oxalic acid dihydrate (Pro analysis), sodium hydroxide (Pro analysis), citric acid monohydrate (Pro analysis), sodium citrate tribasic dihydrate (Pro analysis), and methanol (hypergrade for LC-MS) were all obtained from Merck (Darmstadt, Germany). Accurel PP 1E (R/P) polypropylene flat membrane (100 mm in thickness, 0.1 mm pore size, unknown porosity) was obtained from Membrana (Wuppertal, Germany). The standard 10–1000 mL Biohit tips were from Sartorius Biohit Liquid Handling Oy (Helsinki, Finland), and the Eppendorf safe-lock 2.0 mL PP tubes were produced by Eppendorf AG (Hamburg, Germany). The platinum wires with a diameter of 0.5 mm were obtained from K. A. Rasmussen (Hamar, Norway). 2.2. Preparation of solutions The individual stock solutions of the model peptides were prepared by dissolving the model peptide into deionized water with a concentration of 1 or 2 mg/mL. All these stock solutions were stored at  32 1C and protected from light [26,28]. The sample for the extraction process (step 1) was obtained daily by diluting the stock solutions with 25 mM phosphate buffer with a pH of 3.50. The solution for the optimization of the clean-up process (step 2) was obtained daily by diluting the stock solutions with 75 mM acetate buffer of different pH values. The solution for the clean-up process to evaluate the real performance (combining both extraction and clean-up process) was obtained by adjusting the pH of the acceptor solution after the extraction process to pH 5.25 with an equal volume of 250 mM acetate buffer. 2.3. EME set-up The work flow of the current EME approach is illustrated in Fig. 1, where two pieces of the flat membrane-based EME devices were used. The home-made flat membrane-based EME device has been described elsewhere [27]. An ES 0300-0.45 power supply from Delta Elektronika BV (Zierikzee, Netherlands) was employed, meanwhile the system-current was recorded via a home-made current monitor using LabVIEW 8.2 software (National Instruments, Austin, TX, USA) [28]. The mechanism of cationic peptide transfer by SLM is illustrated in Fig. 1. The negatively charged DEHP at the sample/SLM interface formed complexes with cationic peptides, which facilitated the mass transfer of the cationic peptides from the sample into the SLM/acceptor [28]. The extraction process (step 1) was carried out for 45 min, with a voltage of 15 V, with an SLM of 10 mL of 1-nonanol/2-decanone containing 15% DEHP [28], and under agitation at 900 rpm. In step 1, the sample volume was 600 mL, pH in the sample was adjusted to 3.50 as mentioned in Section 2.2, and 600 mL 50 mM phosphoric acid was used as acceptor solution. Immediately after the termination

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Fig. 1. Work flow of the two-step approach for selective extraction of the target peptide (AT2 AP) using EME (above) (EME for step 1 (extraction process) was performed using a voltage of 15 V for 45 min with an SLM of 1-nanonol/2-decanone (1/1) containing 15% DEHP. EME for step 2 (clean-up process) was performed using a voltage of 5 V for 10 min with an SLM of 1-dodecanol containing 15% DEHP); real picture of the EME device (lower left); and schematic illustration of the mechanism of cationic peptide transferred by the SLM (lower right, reprinted from Ref. [28], copyright @ elsevier).

of step 1, 300 mL of the acceptor solution was collected and transferred to the sample compartment of the second EME device, and 300 mL of 250 mM acetate buffer was added to adjust the pH to 5.25. Meanwhile, 20 mL of the acceptor solution from step 1 was analyzed by HPLC-UV to measure the extraction recoveries during step 1. The clean-up process (step 2) was carried out from the pH adjusted acceptor solution (pH 5.25) for 10 min, with a voltage of 10 V, with an SLM of 10 mL of dodecanol containing 15% DEHP, and under agitation at 900 rpm. The anode was located in the pH adjusted acceptor solution, and 600 mL of 50 mM phosphoric acid was used as waste solution of the other side of the SLM. After 10 min EME in step 2, the pH adjusted acceptor solution was collected and analyzed by HPLC-UV.

2.4. Characterization 2.4.1. HPLC-UV The HPLC-UV analysis was carried out using a Dionex Ultimate 3000 system comprising a pump (HPG-3200M), a degasser (SRD3200), a column oven (FLM-3100), an auto sampler (WPS-3000SL), and a VWD-3400 UV/vis detector operated at 214 nm (all from Dionex Corporation, Sunnyvale, CA, USA). Data were collected and processed using the Chromeleon software (v. 6.80 SP2 Build 2212) from Dionex Corporation. Separations were performed with a Jupiter Proteo C18 column (150 mm  2.00 mm, 4 mm, 90 Å, Phenomenex, Torrance, CA, USA) at 60 1C with an injection volume of 20 mL. Mobile phase A was 20 mM formic acid containing 5% of

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methanol (v/v), and mobile phase B was methanol containing 5% of 20 mM formic acid (v/v). Mobile phase B was increased from 5 to 30% within 8 min at a flow rate of 0.4 mL/min. Afterwards, mobile phase B was further increased to 80% within 0.1 min, and this condition was kept for 2 min before re-equilibration.

2.4.2. pH measurement The pH of the sample and acceptor solution before and after the EME was measured using a 744 pH meter with a microelectrode from Metrohm (Metrohm AG, Switzerland).

2.4.3. Recovery calculation The analyte recovery (Rec) after the extraction process (step 1) and after the extraction þ clean-up process (step 1 þstep 2) was obtained for each peptide using the following equation: Rec ¼ ½C Ai  V A =½C Di ð0Þ  V D   100%

3.2. Extraction process (step 1) The pKa-value of DEHP is 3.50 [35]. The pH in the sample was adjusted to 3.50 based on previous experience to partly ionize DEHP and to obtain efficient mass transfer of peptides into the SLM [28]. The pH value of the acceptor solution was adjusted to 1.80. Low pH conditions were mandatory in the acceptor solution during step 1 for efficient transfer of peptides from the SLM [28]. Thus, the extraction of peptides was performed from 600 mL sample (pH 3.50) to 600 mL of the acceptor solution pH 1.80 for 45 min with a voltage of 15 V. Under these conditions, the obtained recoveries for AT2 AP, AT2, NT, AT1 and L-Enke were 91, 93, 90, 89 and 84%, respectively (RSDo10%), and these values were consistent with previously reported data under similar conditions [28]. Subsequently, the pH of the acceptor solution from step 1 was adjusted to pH 5.25. This pH value was slightly higher than the pI value for AT2 AP, and this will be discussed in more details below.

ð1Þ

where CAi was the measured final concentration of the peptide in the acceptor solution after the designed process, such as step 1 or (step 1 þstep 2), and CDi(0) was the spiked concentration of the model peptides in the sample. VA and VD were the volumes of the acceptor solution and sample, respectively.

3. Results and discussion 3.1. Basic principle The ultimate goal of the current work was to selectively isolate one target peptide (AT2 AP) from a sample also comprising four matrix peptides based on differences in isoelectric point (pI). Thus, as an ideal performance, the recovery of the target peptide in the final acceptor solution should be 100%, whereas the matrix peptides should not be detected in the same solution. The work flow of the present approach is shown in Fig. 1, and was based on a two-step approach. During the extraction process (step 1), all five peptides were transferred from the sample (pH 3.50) and into the acceptor solution (pH 1.80). The cathode was located in the acceptor solution, and the peptides were extracted as net positively charged species across the SLM. The purpose of step 1 was to isolate the target peptide from the bulk sample solution. However, because also the matrix peptides were extracted in this step, a second step (step 2) was required for clean-up based on isoelectric points. Prior to the clean-up process (step 2), the acceptor solution from step 1 was collected and pH was adjusted to pH 5.25 using 250 mM sodium acetate solution. In step 2, the matrix peptides were removed from the pH adjusted acceptor solution, and extracted into the SLM and the waste solution (pH 1.80) with the cathode located in the waste solution. The pI for all matrix peptides is greater than 5.25, so they were net positively charged species in the pH adjusted acceptor solution and were removed by the electric field after the complex formation with negatively charged DEHP. The target peptide with no net positive charge was unable to form complexes with the negatively charged carrier (DEHP) and remained in the pH adjusted acceptor solution containing the anode. For this fundamental study, angiotensin II antipeptide (AT2 AP), angiotensin II (AT2), neurotensin (NT), angiotensin I (AT1), and Leu-enkephalin (L-Enke) were selected as model peptides and represented a relatively wide span in terms of the pI values (ranged from 5.13 to 9.52). The net charge versus pH for all the peptides is presented in Table 1. AT2 AP was selected as the target analyte (pI¼5.13), while the other four peptides (pI45.13) were considered as matrix peptides.

3.3. Clean-up process (step 2) 3.3.1. pH stability The pH value of the pH adjusted acceptor solution was considered an important factor during the clean-up process (discussed later), and the use of a buffer was mandatory to maintain the pH stable around the pI-value (5.13) during step 2. Therefore, acetate buffer was selected based on the pKa of acetic acid (pKa 4.75), which is close to the target pH, and acetate buffers with different molarity (50, 75, 100, 150, and 200 mM) were tested focusing on both the pH shift and the systemcurrent during EME. The system-current should be as low as possible to avoid excessive electrolysis and bubble formation in the sample and acceptor solution. As shown in Table 2, the pH stability of the buffer and the maximal system-current increased with increasing molarity, and the former observation was consistent with general buffer solution theory. Thus, to balance the pH stability and the systemcurrent, acetate buffer with a molarity of 75 mM was selected as the optimal buffer for further experiments. To keep the two-step EME approach as simple as possible, initially, the clean-up process (step 2) was performed in the same EME device as the extraction process (step 1) by diluting 300 mL of the acceptor solution pH 1.80 with 300 mL of 250 mM sodium acetate solution (to adjust pH to 5.25), and by reversing the polarity of the two electrodes. However, high current (4 100 mA) was recorded, even with a voltage of 5 V. Another disadvantage of using the same EME device for step 2 was that the acceptor solution was continuously in contact with the SLM as it was located above the SLM. Thus partial back-extraction and diffusion into the SLM was possible in between step 1 and 2, and this potentially affected the reliability of the concept. Therefore, we decided to perform the clean-up process (step 2) in a second EME device. Thus, immediately after step 1 was finished, the acceptor solution was transferred to a second EME device. In this device, the acceptor solution was pH adjusted prior to step 2, but still it was not in contact with the SLM due to a small air gap below the SLM. Contact between the pH adjusted acceptor solution and the SLM was first established when step 2 was initiated with agitation of the device and application of the electrical field. This circumvented undesired back-extraction and diffusion of peptides into the SLM. Finally, transfer to a second device enabled optimization of the SLM for step 2 independently of step 1 as discussed below. 3.3.2. SLM solvent With the same SLM as in step 1, high system-current (145 mA) was recorded after 5 min clean-up process. Thus, in a next series of experiments, different organic solvents containing 15% DEHP, including 1-nonanol, decanol mixture of isomers, n-decyl alcohol, 1-undecanol,

C. Huang et al. / Journal of Membrane Science 481 (2015) 115–123

1-dodecanol, 2-octyl-dodecanol, 2-nonanone, 2-decanone, 2-undecanone, 2-nitrophenyl octyl ether, 2,4-dimethyl-1-nitrobenzene, and 1ethyl-2-nitrobenzene were tested to reduce the system-current and to optimize the performance of the clean-up process. The recorded maximal system-current and the remaining amount of peptides in the pH adjusted acceptor solution after a 5 min clean-up process are summarized in Table 3. As seen from the data, the SLM of 1-dodecanol

containing 15% DEHP provided the highest amount of AT2 AP found in the acceptor solution after clean-up. Because a high recovery for AT2 AP was a primary goal of the work, 1-dodecanol (þDEHP) was selected as the final SLM for step 2. However, in comparison with other SLMs, 1dodecanol containing 15% DEHP was not the most efficient SLM for removing the four matrix peptides, and those were still detected in the acceptor solution after the clean-up process. Therefore, additional

Table 1 Sequence, iso-electric point and the plot of net-charge versus pH of the peptides. Peptides

Sequence

Iso-electric pointa

AT2 AP

EGVYVHPV

5.13

AT2

DRVYIHPF

7.76

NT

ρELYENKPRRPYIL

9.52

AT1

DRVYIHPFHL

7.91

119

Net-charge versus pHa

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Table 1 (continued ) Peptides

Sequence

Iso-electric pointa

L-Enke

YGGFL

5.93

a

Net-charge versus pHa

Adapted from www.innovagen.se.

Table 2 The pH shift after the EME (ΔpH), and the maximal system-current (Imax) during the EME from acetate buffer solutions with different molarity. EME was performed with an SLM of 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% DEHP and a voltage of 5 V for 5 min, n ¼3. Buffer solutions

ΔpH

Imax (mA)

50mM 75mM 100mM 150mM 200mM

0.117 0.02 0.107 0.01 0.107 0.01 0.09 7 0.01 0.077 0.01

807 10 907 7 1057 3 1427 16 1877 11

Table 3 The maximal system-current (Imax) during the EME and the peptides found in the solution after the clean-up process (Rec) with different SLMs. EME was performed with 75 mM acetate buffer (pH 5.25) with an SLM of different organic solvent containing 15% DEHP and a voltage of 5 V for 5 min, n¼ 3. SLMs

1-nonanol Decanol mixture isomers n-decyl alcohol 1-undercanol 1-dodecanol 1-octyl-dodecanol 2-nonanone 2-decanone 2-undercanone 2-Nitrophenyl octyl ether 2,4-dimethyl-1-nitrobenzene 1-Ethyl-2-nitrobenzene

Imax (mA)

1087 8 47 1 677 11 607 10 197 4 0 537 7 187 4 97 2 27 1 137 2 117 2

Rec% (RSD %) AT2 AP

AT2

NT

AT1

L-Enke

74 63 82 64 91 57 41 42 48 37 40 38

8 (8) nd 7 (43) 7 (39) 10 (53) nd nd nd nd nd nd nd

6 (72) nd 6 (110) 5 (17) 5 (40) nd nd nd nd nd nd nd

nd 4(20) 5 (31) 8 (12) 6 (37) nd 8(18) nd nd nd nd nd

30 50 63 52 73 54 31 70 72 57 17 41

(11) (8) (8) (9) (1) (11) (5) (14) (14) (22) (5) (6)

operational parameters were investigated, such as the pH in the pH adjusted acceptor solution, the DEHP content in the SLM, the extraction voltage, and the extraction time for the clean-up process as reported below.

3.3.3. Solution pH As mentioned above, the pH in the pH adjusted acceptor solution was an important factor during step 2 since the objective was to isolate the target peptide from the matrix peptides based on the isoelectric point. Thus, the acceptor solution pH was optimized for step 2. The tested pH ranged from 3.78 to 5.80 using 75 mM acetate buffers, and the remaining peptides after this clean-up process versus the acceptor solution pH is illustrated in

(15) (7) (3) (7) (4) (10) (19) (10) (5) (2) (112) (9)

Fig. 2. The results showed that within the tested pH range, pH 5.25 was superior. At this pH value, the amount of AT2 AP remaining in the sample was at maximum, and the level of three of the matrix peptides remaining in the sample was at minimum. As seen from Fig. 2, L-Enke (pI¼ 5.93) was poorly removed in step 2. The explanation for this was that L-Enke has almost no net charge in a broad pH range (pH 4 to pH 8) as shown in the plot of the netcharge versus pH (Table 1), and therefore the electrical field had almost no impact on this peptide. The partial removal of L-Enke during step 2 was mainly due to the passive diffusion. Considering the pH shift after the EME (Table 2), the clean-up process was performed actually in the pH range of 5.15–5.25. This maintained the acceptor solution pH at or slightly above the pI of AT2 AP during the whole clean-up process.

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3.3.4. DEHP content In a next series of experiments, different amounts of DEHP in 1dodecanol were investigated as SLM. The tested concentrations of DEHP were 10, 15, 20, 25, and 30% (v/v). The amount of the different peptides remaining in the acceptor solution versus the content of DEHP is plotted in Fig. 3a. In the examined DEHP range, the amount of peptides remaining in the solution including the target peptide decreased with increasing DEHP content. This supported ion-pair formation between the peptides and DEHP at the acceptor solution (pH 5.25)/SLM interface, and was in

121

Table 4 The amount of the peptides found in the solution after the clean-up process (Rec) with different times. EME was performed with 75 mM acetate buffer (pH 5.25) with an SLM of 1-dodecanol containing 15% DEHP and a voltage of 5 V for different times, n¼ 3. Time (min)

5 10 15 20

Rec (RSD %) AT2 AP

AT2

NT

AT1

L-Enke

87 83 77 74

6 (22) nd nd nd

4 (25) nd nd nd

4 (20) nd nd nd

65 56 42 37

(5) (6) (5) (6)

(5) (6) (4) (16)

100

Rec(%)

80 AT2 AP

60

AT2 NT

40

AT1 20 0 3.75

Enke

4.05

4.35

4.65

4.95

5.25

5.55

5.85

pH Fig. 2. Effect of the solution pH for the clean-up process. EME was performed with 75 mM acetate buffer (pH varied from 3.78 to 5.80) with an SLM of 1-dodecanol containing 15% DEHP and a voltage of 5 V for 5 min, n¼ 3.

Fig. 4. Chromatograms of the solution after the clean-up process (STEP2), the acceptor solution after the extraction process (STEP1) and the standard solution in 25 mM phosphate buffer with a pH of 3.50 (STD)(The elution order is AT2 AP, AT2, NT, AT1 and L-Enke, respectively.). STEP2: EME was performed with the acceptor solution pH 5.25 (obtained by the adjustment of the pH of the acceptor solution after the extraction process with sodium acetate solution) for 10 min with an SLM of 1-dodecanol containing 15% DEHP and a voltage of 10 V. STEP1: EME was performed with an SLM of 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% DEHP and a voltage of 15 V for 45 min from spiked 25 mM phosphate buffer (pH 3.50) to 50 mM phosphoric acid. The UV-detector was operated at the wavelength of 214 nm, and the solution for performing step 2 was diluted twice from the acceptor solution after step 1.

accordance with previous observations [28]. Meanwhile, higher DEHP concentration generated slightly higher system-current in the EME system. Accordingly, concerning the criterion of high amount of AT2 AP and low amount of other matrix peptides remaining in the solution, the SLM of 1-dodecanol containing 15% DEHP was selected as the optimal SLM.

Fig. 3. Effect of the content of DEHP in the SLM (a) and the effect of voltage (b) in the clean-up process. EME was performed with 75 mM acetate buffer (pH 5.25) for 5 min with an SLM of 1-dodecanol containing 10 to 30% DEHP and a voltage of 5 V (a), or with an SLM of 1-dodecanol containing 15 % DEHP and a voltage of 0 to 20 V (b), n ¼3.

3.3.5. Voltage Subsequently, the voltage used during step 2 was optimized, and the examined voltage ranged from 0 to 20 V. The amount of the different peptides remaining in the acceptor solution versus the voltage is plotted in Fig. 3b. AT2 AP and L-Enke were unaffected by the voltage, because the net charge of these peptides was zero (or very close to zero). However, the voltage displayed significant effects on the other three matrix peptides (the inset in Fig. 3b) due to the net positive charges. Higher voltage resulted in higher system-current, and the difference in removal of the charged peptides using 10 V or higher voltage was negligible. According to the above observations, 10 V was selected as the optimal voltage for the clean-up process.

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3.3.6. Extraction time In a final experiment, the clean-up process (step 2) was carried out for 5, 10, 15 and 20 min. The amount of the different peptides remaining in the acceptor solution after the clean-up process for different clean-up times is summarized in Table 4. Obviously, the amount of the different peptides remaining in the acceptor solution decreased with increasing clean-up time. Prolonging the clean-up time from 5 to 10 min showed a small loss of AT2 AP, but improved the removal of matrix peptides significantly. Thus, after the clean-up process was performed for 10 min (or longer), all positively charged matrix peptides were undetectable except LEnke (with close to zero net charge). Thus, 10 min was selected as the clean-up time.

separate EME units was a disadvantage, but from recent work we know it is possible to perform EME in 96-well plates [36], and transfer of the current principle to 96-well technology definitely will improve the work flow. Work is currently in progress also to test the two-step EME concept for biological fluids, and for peptides with higher pI values than the target analyte in the present work. For this work, and for all future work with similar systems by other researchers, the fundamental investigations and experiences reported in the current work should be very important information.

3.4. Performance

This work has been performed as part of the ‘Robust affinity materials for applications in proteomics and diagnostics’ (PEPMIP) project, supported by the Seventh Research Framework Program of the European Commission. Grant agreement number: 264699.

As reported above, for the extraction process (step 1), EME was performed with an SLM of 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% DEHP and a voltage of 15 V for 45 min from spiked 25 mM phosphate buffer (pH 3.50) to 50 mM phosphoric acid, and the recoveries for all five peptides were in the range of 84–93%. For the clean-up process (step 2), EME was carried out with an SLM of 1-dodecanol containing 15% DEHP and a voltage of 10 V for 10 min from spiked acetate buffer (pH 5.25) to the waste solution (50 mM phosphoric acid). To evaluate the total performance of the combination of the extraction process (step 1) and the clean-up process (step 2), the pH of the acceptor solution after the extraction process was adjusted to pH 5.25 prior to the clean-up process. The chromatograms of the standard solution, the acceptor solution after the extraction process (step 1), and the acceptor solution after the clean-up process (step 2) are illustrated in Fig. 4, and these demonstrate that selective isolation of the target peptide from the matrix peptides based on differences in isoelectric point was possible. The amount of AT2 AP remaining in the solution after the clean-up process was found to be 73% with a RSD of 13%. This was considered as a high recovery, although a small fraction of the analyte was lost in the process. The three positively charged peptides AT2, NT and AT1 were not detected in the solution, which showed high separation factors (the ratio between the target peptide recovery and the matrix peptide recoveries) as compared to IEF using EMP [30]. These observations above clearly demonstrate a potential for selective isolation of target peptides from matrix peptides by EME based on differences in isoelectric point. However, 48% L-Enke (with a RSD of 5%) could also be detected after this twostep approach due to the almost zero net charge at pH 5.25 to 5.15 (Table 1), which is consistent with the separation of peptides or proteins by iso-electric focusing [31,32]. Complete removal of L-Enke was not obtained in the current paper, but this is currently under fundamental investigation.

4. Conclusions The present fundamental study has for the first time demonstrated selective extraction of peptide based on differences in isoelectric point using a two-step EME approach. After two-step EME, 73% AT2 AP was found in the solution, which indicates that two-step EME can be used for removal of peptides based on major differences in isoelectric point, and this very interesting aspect should definitely be investigated in more detail for future applications. The experimental data also suggested that separation of peptides with small differences in isoelectric point may be more challenging. The current work utilized two separate EME units to perform step 1 and 2, respectively. This provided high flexibility and enabled independent optimization of the extraction step and the clean-up step. In terms of work flow, however, the use of two

Acknowledgment

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