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Theresa Kristl Hanno Stutz
Research Article
Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants†
Received September 1, 2014 Revised October 20, 2014 Accepted October 20, 2014
One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1–10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3–10 with Pharmalyte 5–6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5–6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. L-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. L-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed. Keywords: Capillary isoelectric focusing / Chemical mobilization / Food allergens / Ovalbumin / Pressure mobilization DOI 10.1002/jssc.201400890
1 Introduction The characterization of proteins, for example, by product profiling, constitutes an important aspect of quality assurance. In biological and biopharmaceutical products, for example, (recombinant) allergens, post-translational modifications (PTMs) but also process- and product-related impurities [1] increase the number of protein species and thus the product complexity. In this context, CE has been applied to separate closely related allergen variants [2–4]. Food allergens affect up to 8% of children and represent a key source of allergic disorders [5]. Ovalbumin (OVA), an albumin species most abundant in egg white [6], is a major causative elicitor of food allergies [7] encountered not only in eggs and related products, but also in processed food [8] where egg white is added to provide additional nutrients and improve flavor or texture [9]. Food with OVA contamination or without appropriate declaration are thus detrimental for allergic patients [10]. Moreover, OVA is also present in Correspondence: Dr. Hanno Stutz, Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria E-mail: [email protected] Fax: +43-662-8044-5751
Abbreviations: CA, carrier ampholyte; Ca II, carbonic anhydrase isoform II; IDA, iminodiacetic acid; OVA, ovalbumin; PL, Pharmalyte; PTM, post-translational modification C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vaccines produced in chicken eggs and shows content fluctuations in seasonal vaccine batches [11]. In addition, OVA is used as a prominent allergen frequently applied for sensitization and challenging experiments in immunological studies [12, 13], for example, in asthma mouse models [14]. OVA is composed of 385 amino acids with 42.9 kDa, possesses one acetylation, two phosphorylation sites, and an N-glycosylation at Asn292 [15, 16]. Different occupations of acetylation and phosphorylation sites influence the pI. Recently, an additional PTM decoration, that is, by nitration, was shown to enhance the allergenicity of modified OVA [17]. Moreover, commercial OVA products were shown to contain copurified minor impurities [16]. The manifold applications of OVA emphasize the importance of an appropriate standardization and profiling of commercial products that are used in immunological studies. A cross-evaluation of applied OVA products proving equivalency or revealing differences is thus supportive for the comparability of immunological results. Within the arsenal of bioanalytical techniques for distinction of intact proteins and PTMs, CE has conquered a distinguished position due to its outstanding separation efficiency. Among its different modes, particularly CIEF is considered to provide outstanding selectivity [18–21]. The separation † This paper is included in the virtual special issue on Amino acids, proteins and peptides available at the Journal of Separation Science website.
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principle addresses differences of pI according to Eq. (1) for resolution in (C)IEF: D.[d(pH)/dx] ⌬(pI) = 3 (1) E .[−d/d(pH)] and theoretical differences down to ⌬0.02 pI units can be resolved [22]. Thereby, D represents the diffusion coefficient of the analyte, d(pH)/dx is the spatial change of pH within the generated gradient, −d/d(pH) is the mobility change with pH at the respective pI, and E is the electric field strength. The fundamentals of CIEF within a pH gradient formed by commercial carrier ampholytes (CAs) have been reviewed comprehensively [23–25]. The primary pH gradient generated by the applied anolyte and catholyte solutions is stabilized by CAs [26]. Since the quality of CAs varies [27, 28] and will influence the separation performance, an appropriate CA selection and blending different CAs that cover different pH domains have also proven their importance in establishing selectivity. This can be achieved by a local widening of the gradient within the pH domain that covers the pI of target analytes [3, 29]. However, composition of the CA mixture is delicate to prevent extended focusing caused by analyte retardation due to progressive loss in net charge when approaching its focusing site. This might result in incomplete focusing [22, 30]. Moreover, analyte focusing in the distal capillary part behind the detector, and time-dependent changes of the pH gradient by ITP-driven losses of highly acidic and alkaline CAs at the capillary ends are essential limitations [31,32]. Reagents that block the very acidic and alkaline sections, that is, so-called spacer, counteract these drawbacks [29–31, 33], but have to be adjusted to the respective separation challenge to fulfill their task without compromising the resolution by compressing the pH gradient via substantial occupation of the terminal capillary parts. Different mobilization strategies have been developed and applied in CIEF, including mobilization by pressure and chemical means [34]. Although advantages and disadvantages of the respective strategies have been discussed, a competitive comparison of mobilization approaches for the same analyte implementing an individual tuning of CAs is still neglected. Since OVA with its various PTMs and concomitant subtle differences in the pI can be considered a challenging analyte, differences in the potential of the respective mobilization strategies will become evident. This work targets to provide a comparative evaluation of mobilization strategies for two-step CIEF. From a common CIEF basis, individual mobilization strategies are set and optimized followed by a subsequent refinement of the CIEF focusing step, respectively. The most distinguished CIEF method will then be employed for profiling and comparison of various commercial OVA products.
2 Materials and methods 2.1 CE Measurements were performed on the Agilent 7100 Capillary Electrophoresis System (Agilent Technologies, Waldbronn, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Germany). The 7100 CE system was equipped with a diodearray detector covering 190–600 nm. For cutting-off low wavelengths that would interfere with absorbance of CAs, detector filter assembly G7100–62700 (from Agilent Technologies) was used for executing CIEF at 270 nm. Band width and scan rate were set to 4.0 nm and 2.5 Hz, respectively. Data treatment was done with Chem Station, Rev. B.04.03. For CIEF separation, eCAPTM neutral coated CIEF capillaries (Beckman CoulterTM , La Brea, CA, USA) with 50 m id, effective capillary length to detector (LD ) 23.0–23.9 cm, and total capillary length (LT ) 31.4–32.6 cm were aligned in the interface for 50 m straight capillaries. The cassette temperature was set to 20.0⬚C. The current limit was selected with 20 A according to the recommendation of the capillary manufacturer to protect the capillary coating.
2.2 Chemicals Sodium hydroxide (w = 32%), orthophosphoric acid (H3 PO4 , w = 85%), and acetic acid (glacial; 100%) were obtained from Merck (Darmstadt, Germany). L-Arginine (L-Arg) and iminodiacetic acid (IDA)—used as spacer solutions— and L-glutamic acid (L-Glu; for chemical mobilization) were from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was prepared by a Milli-Q Plus 185 system (Millipore, Molsheim, France). The cIEF gel was acquired from Beckman Coulter. CAs PharmalyteTM pH 3–10 and PharmalyteTM pH 5–6 were obtained from GE Healthcare (Piscataway, NJ, USA). Anolyte and catholyte stock solutions were 1.00 mol/L H3 PO4 and 1.00 mol/L NaOH, respectively. Stock solutions of cathodic and anodic spacer were 500 mmol/L L-Arg and 200 mmol/L IDA, respectively, as outlined elsewhere [33]. For chemical mobilization different stock solutions were prepared, including 2.20 mol/L acetic acid, and 50 mmol/L L-Glu. The pH of 50 mmol/L L-Glu was either adjusted to 10.50 with 1.00 mol/L NaOH for application with normal pH gradient (acidic domain at capillary inlet), or to 2.15 by adding 85% and 1.00 mol/L H3 PO4 when applied in the reversed pH gradient, respectively. All stock solutions were stored between +4 and +8⬚C and proved stability for at least 4–5 months under these conditions.
2.3 Proteins and pI markers Albumin from chicken egg white (OVA) was purchased from Sigma-Aldrich in different purities specified as grade II (crude dried egg white 62–88% OVA), grade V (ࣙ98% purity), and grade VI (ࣙ98% purity and low mannose content) by the manufacturer. Stock solutions of OVA were prepared in ultrapure water at 5.83 mg/mL (grade II), 16.75 mg/mL (grade V), and 2.52 mg/mL (grade VI). Solutions of OVA grade II and VI were filtered due to slight precipitates. Myoglobin from horse heart (min. 90%) and carbonic anhydrase isoform II (Ca II from bovine erythrocytes, pI 5.91 [35] and 5.4) were obtained from Sigma-Aldrich. Peptide markers with pI 10.0, 9.5, www.jss-journal.com
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7.0, 5.5, and 4.1 and primary sequence according to Shimura et al. [35], and ribonuclease A (pI 9.68 [35]) were obtained from Beckman Coulter. Synthetic peptides with pI 4.72 (P1) and pI 4.51 (P2) were synthesized on demand with a purity >95% by Metabion International AG (Martinsried, Germany). The pI values of these peptides have been tailored by the authors via selection of appropriate amino acid sequences to flank OVA variants as closely as possible. Stock solutions of horse heart myoglobin (1.00 mg/mL), ribonuclease A (24.00 mg/mL), Ca II (pI 5.4; 4.06 mg/mL), Ca II (pI 5.91; 4.00 mg/mL), P1 (0.95 mg/mL), and P2 (1.00 mg/mL) were prepared in ultrapure water. Aliquots of 25 L were prepared from stock solutions and stored at −20⬚C. After thawing, aliquots were rapidly consumed. CIEF samples contained 81.5% v/v cIEF gel, spacer compounds in appropriate concentrations, model peptides and proteins, and OVA. The sample composition varied and details are given in the respective sections and figure captions.
2.4.1.2 Chemical mobilization with acetic acid or glutamic acid (pH 10.50) CIEF was performed with 200 mmol/L H3 PO4 in cIEF gel (anolyte) and 300 mmol/L NaOH (catholyte) [33] applying +25.0 kV for 15.0 min. For chemical mobilization the catholyte was either replaced with (i) 22 mmol/L acetic acid or (ii) 50 mmol/L L-Glu (adjusted to pH 10.50) with application of +30.0 kV [36].
2.4 CIEF
3 Results and discussion
Pretreatment of eCAP neutral capillaries is outlined elsewhere [3]. Briefly, new eCAP capillaries were conditioned with 10 mmol/L H3 PO4 for 4 min followed by ultrapure water for 15 min (900 mbar each). Before the first daily CIEF separation, the capillary was flushed with ultrapure water for 15 min (900 mbar). Before each CIEF run, the eCAP capillary was flushed with 90.9 mmol/L H3 PO4 in cIEF gel for 2 min followed by ultrapure water for 4 min (both 900 mbar). Anolyte, catholyte, and sample solutions were prepared freshly and vortexed thoroughly followed by a centrifugation step with 18 600 × g for 5 min at 20⬚C to remove air bubbles and particulate matter before their use. Samples were injected hydrodynamically with 900 mbar for 200 s to assure filling of the entire capillary with the sample. Capillary ends were dipped into ultrapure water after injection to avoid carryover. Settings for subsequent focusing and the optimized mobilization steps are specified in Section 2.4.1. Anolyte and catholyte solutions were used for two to four runs depending on CIEF analysis time to prevent electrolyte depletion. After each CIEF run, the capillary was flushed with ultrapure water for 4 min (900 mbar). Daily measurements were finished by filling the eCAP capillary with cIEF gel and subsequent storage at +4 to +8⬚C with capillary ends dipped into ultrapure water.
3.1 Optimization of spacer concentrations
2.4.1 Focusing and mobilization 2.4.1.1 Pressure mobilization During CIEF focusing, +15.0 kV were applied for 6 min with 90.9 mmol/L H3 PO4 in cIEF gel as anolyte and 19.2 mmol/L NaOH (in water) as catholyte. During subsequent pressure mobilization 48 mbar were applied on the anodic site with auxiliary +21.0 kV. Focusing setting is slightly different from strategies using chemical mobilization due to baseline irregularities. Details are given in the text. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4.1.3 Chemical mobilization with reversed pH gradient and glutamic acid (pH 2.15) Positions of anolyte (200 mmol/L H3 PO4 in cIEF gel) and catholyte (300 mmol/L NaOH in water) [33] were changed thus reversing the pH gradient with the acidic domain situated at the detector side. Focusing was done at −25.0 kV for 15.0 min. For mobilization, the anolyte was exchanged against 50 mmol/L L-Glu (pH 2.15) with application of −30.0 kV.
The implementation of spacer compounds in the CIEF sample ensures temporal stabilization of the pH gradient by preventing CA losses to electrolyte vessels. This contributes to an enhanced repeatability of mobilization times [33, 37]. Efficient spacer compounds should ideally act as good CAs [23] and additionally occupy the pI gap between most acidic CAs and the anolyte and most alkaline CAs and the catholyte, respectively [33]. Therefore, L-Arg (pI 10.76) [22] and IDA (pI 2.23) [38] were selected as cathodic and anodic spacer compounds as described recently [32,33]. Concentrations and thus zone lengths of spacer can be selected individually since IDA has to overcome anodic losses of CAs, whereas L-Arg prevents cathodic CA losses and may additionally block the distal capillary part behind the detector according to requirements. 3.1.1 Spacer concentrations with pressure mobilization Different spacer concentrations were tested with pressure mobilization to reveal their effects on selectivity and detection, applying a mixture of model proteins/peptides and a preoptimized CA mixture of 0.8% m/v Pharmalyte 3–10 (PL 3–10) and 0.5% m/v Pharmalyte 5–6 (PL 5–6; Fig. 1). L-Arg was tested at 0, 10.7, 21.4, and 42.9 mmol/L with highest concentrations referring to a previous test series [33] in combination with 0.4–1.4 mmol/L IDA. Without spacer addition, intensities of peaks presumed to represent alkaline and neutral analytes were too low for an unambiguous assignment (data not shown). Increased L-Arg concentrations progressively enhanced signal intensities primarily in the alkaline domain that also explains visibility of so-called cathodic peaks at higher L-Arg concentrations (Fig. 1, traces a–c). Both effects www.jss-journal.com
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Figure 1. Influence of spacer concentrations on the CIEF separation with pressure mobilization. The sample contained 0.8% m/v PL 3–10, 0.5% m/v PL 5–6, 0.43 mg/mL ribonuclease A (RnA), 0.038 mg/mL horse heart myoglobin (HHM), 0.036 mg/mL Ca II (5.91), 0.045 mg/mL Ca II (5.4), 10.15 g/mL P1, 5.35 g/mL P2, and cIEF gel, including the following spacer combinations: (a) 10.7 mmol/L L-Arg + 0.4 mmol/L IDA, (b) 21.4 mmol/L L-Arg + 0.9 mmol/L IDA, (c) 42.9 mmol/L L-Arg + 1.4 mmol/L IDA. Anolyte: 90.9 mmol/L H3 PO4 in cIEF gel; catholyte: 19.2 mmol/L NaOH. Either sample was focused for 6 min at +15.0 kV. Mobilization was done by 48 mbar at +21.0 kV. Arrows indicate region of baseline irregularities.
can be related to a progressive compression of the pH gradient by the spacer. Cathodic peaks occur due to a transient bidirectional head-to-head movement of analytes from either capillary end toward their focusing position [30, 31, 33]. In the alkaline to neutral pH region, the resolution improved, whereas it decreased in the acidic region when L-Arg concentration was raised. This discrepancy as well as the prominent increase in peak heights in the region between pH 6 and 10 might be related to a nonuniform compression of the pH gradient, whereby the acidic part that additionally contains PL 5–6 is less affected. However, currently it is not clear without ambiguity whether alkaline and neutral peaks at 10.7 mmol/L L-Arg represent focused zones or anodic peaks on their way toward the final focusing site behind the detector that are prematurely mobilized (Fig. 1, trace a). The increase to 21.4 and 42.9 mmol/L L-Arg would shift the focusing position in front of the detector (compare results of Section 3.1.2). This would offer an alternative explanation for improved resolution and increased peak heights (Fig. 1, traces a–c), since compression of the pH gradient would accelerate focusing, assure its completion, and result in increased peak heights. In parallel, the combined focusing and mobilization time (tm ) of model proteins and peptides increased due to the accretive occupation of the distal capillary part by L-Arg and the concomitant increase in distance that analyte zones are focused ahead of the detector. Remarkably, baseline irregularities occurred within the focusing region of OVA (pI 4.51–4.72) when increasing L-Arg (Fig. 1, traces a–c). These baseline irregularities are apparently related to the applied wide pH range CA, that is, PL 3–10, in interplay with L-Arg concentration. Concentration reduction of both PL 3–10 and L-Arg attenuated baseline irregularities. The described effects were duplicated with different batches of PL 3–10 and also by replacing L-Arg with other cathodic spacers, that is, L-Lys or N,N,N ,N -tetramethylethane1,2-diamine (TEMED) [22, 39] (data not shown). Moreover, an increase in the focusing time, for example, to 15 min, also increased background fluctuations. Evidently, a slightly unequal distribution of CA species within this pH regime
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together with the compression of pH gradient could yield the baseline unsteadiness. No comparable effects were encountered for IDA up to the highest concentration tested, that is, 10 mmol/L (data not shown). Irrespective of the proposed explanatory model, low L-Arg concentrations would favor improved resolution in the acidic domain and thus of OVA and reduce the UV background at the expense to miss alkaline constituents. 3.1.2 Chemical mobilization The effects of spacer and CA described in the previous section could not be transmitted a priori to chemical mobilization with acetic acid. Thus, testing was repeated but confined to L-Arg concentrations of 10.7, 21.4, 42.9 mmol/L, maintaining the IDA concentration at 1.8 mmol/L, respectively. Contrary to the pressure mobilization, only 1.8% m/v PL 3–10 without PL 5–6 was applied. The increase in peak heights of alkaline to neutral marker was not observed when rising L-Arg. This seems to corroborate the derived explanatory model considering also that PL 5–6 was not included. However, L-Arg had to exceed 20 mmol/L to allow for the detection of marker compounds with pI 10.0 and 9.5 by sufficient occupation of the capillary blind end as corroborated by recent results [33]. Although the cathodic spacer slightly increased the baseline noise, pronounced irregularities as in pressure mobilization were absent except for a small rectangular step at pI < 4.1 that will not interfere with OVA (data not shown).
3.2 Optimization of CA composition for different mobilization strategies 3.2.1 Pressure mobilization After the selection of spacer concentrations, different combinations and concentrations of wide and narrow pH range
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J. Sep. Sci. 2015, 38, 148–156 Figure 2. Optimization of CIEF with pressure mobilization for OVA (grade V) by various CA combinations all given as m/v. (A) Resolution improvement by continuous reduction of PL 5–6 maintaining 0.8% PL 3–10 throughout all CIEF experiments. Traces: (a) 1.5% PL 5–6, (b) 1.0% PL 5–6, (c) 0.3% PL 5–6. (B) Resolution improvement of OVA fractions by variation of PL 3–10 maintaining 0.5% PL 5–6 throughout all CIEF experiments. Traces: (a) 1.5% PL 3–10, (b) 0.8% PL 3–10, (c) 0.3% PL 3–10. (C) CIEF separation with optimized composition of wide and narrow range CAs. Sample: 0.3% PL 3–10, 1.0% PL 5–6, 0.36 mg/mL OVA (grade V) with (a) 10.7 mmol/L L-Arg and 1.8 mmol/L IDA, (b) no spacer added. Signal fractions of OVA are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
CAs were tested to optimize the pH gradient in the focusing region of OVA. Based on an initial preoptimization, a combination of 0.8% m/v PL 3–10 and 0.5% m/v PL 5–6 (see Section 3.1.1) was selected as starting point. During the optimization procedure, one CA was maintained and the other varied. When PL 3–10 was kept at 0.8% m/v, PL 5–6 was varied in six incremental steps from 0.3 to 2.0% m/v, selecting 10.7 mmol/L L-Arg and 1.8 mmol/L IDA as spacer. L-Arg was slightly reduced to lower baseline signals and improve resolution since OVA is focused on the capillary inlet part. Best resolution of OVA was achieved with 0.8% m/v PL 3–10 and 1.0% m/v PL 5–6 (Fig. 2A) with three major peaks (peaks 2–4), a minor peak (peak 6), and an additional shoulder at the most acidic major OVA fraction (peak 5). Lower and higher PL 5–6 concentrations progressively deteriorated resolution (Fig. 2A, traces a–c). Subsequent optimization addressed the variation of PL 3–10 between 0.3 and 1.8% m/v, keeping the original PL 5–6 content, that is, 0.5% m/v, revealing best resolution at 0.3–0.5% m/v PL 3–10 (Fig. 2B, traces a–c). The low PL 3–10 concentration favorably reduces the baseline irregularities in the OVA domain. A final optimization selecting 0.3% m/v PL 3–10 with further testing of PL 5–6 closely around the previously optimized 0.8% revealed a slightly higher PL 5–6 content more advantageous. Thus, the finally optimized CA combination comprised 0.3% PL 3–10 with 1.0% (all m/v) PL 5–6 in combination with 10.7 mmol/L L-Arg and 1.8 mmol/L IDA (Fig. 2C, trace a). When OVA was analyzed with this CA combination, but in absence of spacer, resolution was substantially improved (Fig. 2C, trace b). This is due to an expansion of the pH gradient and the reduction of the preponderant contribution of the described baseline anomaly that interferes with OVA. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2.2 Chemical mobilization with acetic acid For chemical mobilization with a weak acid, that is, acetic acid, initial testing was confined to PL 3–10 with 1.8% m/v. Mobilization was done by replacing the catholyte with acetic acid during the mobilization step. Concentrations between 91.0 and 200 mmol/L H3 PO4 (anolyte) and between 18.9 and 300 mmol/L NaOH (catholyte), respectively, showed no substantial difference in resolution and peak shapes of model peptides (pI 4.1–10.0). Therefore, the highest tested anolyte and catholyte concentrations were kept for repeatability reasons [33]. Moreover, a stepwise increase of the acetic acid concentration in the mobilizer solution from 22 to 175 mmol/L showed no difference in the separation result and thus 22 mmol/L was selected. All further CIEF measurements were performed with spacer concentrations optimized for pressure mobilization (see Section 3.2.1), that is, 10.7 mmol/L L-Arg, 1.8 mmol/L IDA. CA optimization was started by stepwise increase of PL 3–10 from 1.2 to 2.8% m/v (without PL 5–6; data not shown). The best PL 3–10 concentration, that is, 1.8%, was kept and then incrementally supplemented with 0.3–2.5% m/v PL 5–6. Resolution of OVA fractions improved up to 1.5% PL 5–6 and remained virtually unaffected by higher PL 5–6 concentrations (Fig. 3A, traces a–c). This is related to a pronounced capillary occupation of pH 5–6 section and adjacent parts within the pH gradient, which causes disappearance of peptide pI 7.0 starting with 2.0% m/v PL 5–6 due to its focusing behind the detector. With 1.5% m/v PL 5–6, this peptide occurred again within the focusing step. In parallel, peptide pI 5.5 started to split in two separate peaks at 0.8% m/v PL 5–6, with increasing spatial distance between these twin peaks at higher PL 5–6 concentrations (data not www.jss-journal.com
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Figure 3. Optimization of CAs (all given as m/v) for chemical mobilization with 22 mmol/L acetic acid. Details of CIEF separation depicted for the focusing domain of OVA (grade V). (A) Resolution improvement of OVA fractions by variation of PL 5–6 with 1.8% PL 3–10 throughout all CIEF experiments. Traces: (a) 0.3% PL 5–6, (b) 1.5% PL 5–6, (c) 2.5% PL 5–6. (B) Resolution improvement by variation of PL 3–10 maintaining 1.5% PL 5–6. Traces: (a) 0.5% PL 3–10, (b) 0.8% PL 3–10, (c) 2.8% PL 3–10. Separated fractions are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
shown). In a second approach the optimized content of 1.5% m/v PL was kept with variation of PL 3–10 between 0.5 and 2.8% m/v. The combination of 0.8% PL 3–10 and 1.5% m/v PL 5–6 provided best resolution (Fig. 3B, traces a–c) with separation pattern nearly congruent to 1.8% PL 3–10 and 1.5% m/v PL 5–6, but at faster tm (Fig. 3A and B, traces b, respectively).
3.2.2.1 Signal splitting The described peak splitting of peptide pI 5.5 (see Section 3.2.2) has been related to insufficient focusing under the selected conditions. Increasing the relative PL 5–6 content from 0.8 to 2.5% in the CA mixture, keeping 1.8% (all m/v) PL 3–10 progressively enhanced the time interval between pI 5.5 twin peaks, whereas an increase in the portion of PL 3–10 (from 0.5 to 1.8% m/v with constant PL 5–6) reduced their time difference without achieving complete mergence. Sole operation with PL 3–10 ceased the splitting effect (data not shown). This phenomenon can be explained by the continuous retardation in velocity during the focusing step due to a concomitant reduction in net charge when approaching the focusing region [22]. For synthetic peptides (pI 4.72 and 4.51) flanking OVA, these effects were absent thus ruling out contribution of artificial peak splitting to the observed OVA heterogeneity (data not shown). This justifies also the application of a narrow pH range CA with a slight offset from the pI of OVA. Since CA species extent over their nominal range [3, 40], their leakage in the pI region of interest will improve the resolution by an appropriate flattening of the pH slope without requiring extended C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
focusing times that inherently also bear the risk of protein precipitation. 3.2.3 Chemical mobilization with zwitterion Alternatively, a chemical mobilization with a zwitterion, that is, 50 mmol/L L-Glu, was tested according to ref. [36]. Therefore, the pI of the mobilizing zwitterion (L-Glu pI = 3.22) [41] was selected below that of OVA to assure focusing of L-Glu behind the analyte and thus a mobilization of the analyte toward the detector. The spacer concentrations and initially also the CA combination that had been optimized for acetic acid were both maintained. Alike for acetic acid, baseline noise in the focusing region showed only a tiny increase compared to the overall baseline. Again, the CA composition was refined varying PL 3–10 between 0.5 and 1.2%, while maintaining 1.5% (all m/v) PL 5–6 (Fig. 4A, traces a–c). Subsequently, PL 5–6 was varied between 1.0 and 2.0% (Fig. 4B, traces a–c) with 0.8% (all m/v) PL 3–10 retained as most favorable as evident from the previous optimization (Fig. 4A, trace b). The combination of 0.8% PL 3–10 and 2.0% m/v PL 5–6 provided best selectivity with resolution of two major and ten minor peaks (Fig. 4B, trace c). 3.2.4 Chemical mobilization with zwitterion and reversed pH gradient Due to a total CIEF analysis time (i.e., focusing and mobilization) of approximately 33 min (Fig. 4B, trace c), acceleration was aimed by a reversal of anolyte and catholyte, and thus the pH gradient (acidic part at capillary outlet; see www.jss-journal.com
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Figure 4. Resolution improvement of OVA (grade V) via optimization of CA composition (all given as m/v) for chemical mobilization with 50 mmol/L L-Glu (pH 10.50). (A) Variation of PL 3–10 maintaining 1.5% PL 5–6. Traces: (a) 0.5% PL 3– 10, (b) 0.8% PL 3–10, (c) 1.2% PL 3–10. (B) Variation of PL 5–6 maintaining 0.8% PL 3–10. Traces: (a) 1.0% PL 5–6, (b) 1.5% PL 5–6, (c) 2.0% PL 5–6. Separated fractions are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
Section 2.4.1.3). The idea is to focus OVA intentionally behind the detector in the blind part of the capillary requiring subsequent mobilization toward the capillary inlet with OVA zones approaching the detector from its rear. This will substantially reduce the required mobilization time. The CA combination from Section 3.2.3 was maintained, including only IDA, but not L-Arg. Indeed, the mobilization time was considerably accelerated but some minor fractions were already detected during focusing. In total, four fractions were distinguished. When the focusing time was increased from 10 to 20 min to exclude presence of artifact peaks, the peak pattern remained unchanged excluding insufficient focusing (data not shown). To avoid focusing within the detection window, L-Arg was incrementally added (0–36.6 mmol/L), whereas IDA was omitted. With 36.6 mmol/L L-Arg, OVA was entirely pushed to the blind capillary end and CIEF analysis time was 24 min (Fig. 5A and B). However, selectivity was impaired and similarly to pressure mobilization baseline irregularities that interfered with some OVA fractions were introduced.
3.3 Profiling of commercial OVA products Based on the results of the optimized CIEF mobilization strategies, chemical mobilization with 50 mmol/L L-Glu pH 10.50 was selected most appropriate and applied for profiling different commercial OVA products, specified as grade II, V, and VI (Fig. 6, traces a–c). Grade V and VI showed only marginal differences as expected from the specifications of the manufacturers (see Section 2.3). Two major fractions (peaks 5 and 8, Fig. 6) and 8–10 minor fractions were separated (Fig. 6). The relative abundance of the major peaks C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. CIEF optimization for chemical mobilization with 50 mmol/L L-Glu (pH 2.15) with reversed pH gradient. (A) Application of different L-Arg concentrations: (a) no L-Arg, (b) 10.7 mmol/L L-Arg, (c) 21.4 mmol/L L-Arg, (d) 36.6 mmol/L L-Arg. The sample contained 0.8% m/v PL 3–10, 2.0% m/v PL 5–6, 0.36 mg/mL OVA (grade V), 5.35 g/mL P2 (pI 4.51) and cIEF gel. The sample was focused for 15 min at −25.0 kV. Mobilization was done by replacing the anolyte with 50 mmol/L L-Glu (pH 2.15) and application of −30.0 kV. (B) Detail of the optimized CIEF separation pattern with 36.6 mmol/L L-Arg.
was nearly congruent for both products. In case of grade II, the alkaline major fraction (peak 5) was considerably reduced, whereas acidic variants (peaks 9 and 10) were more prominent (Fig. 6, trace a).
4 Concluding remarks A comprehensive comparison of different mobilization strategies for a major food allergen, that is, OVA, has been www.jss-journal.com
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constituent profile of three commercial OVA products and proved the suitability of the method for the outlined aim. Agilent Technologies (Waldbronn, Germany) is kindly acknowledged for providing an Agilent 7100 CE System as a loan in the course of a Research Sponsoring Agreement. The authors are indebted to Dr. Gerard Rozing, Dr. Martin Greiner, and Dr. Hans-Josef Brunnert (all from Agilent Technologies, Waldbronn) for their support within the research cooperation. The authors have declared no conflict of interest.
5 References [1] ICH Steering Committee, ICH-Guideline Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products 1999, pp. 1–16. [2] Punzet, M., Ferreira, F., Briza, P., van Ree, R., Malissa, H., Stutz, H., J. Chromatogr. B 2006, 839, 19–29. [3] Kronsteiner, B., Malissa, H., Stutz, H., Electrophoresis 2007, 28, 2241–2251.
Figure 6. Testing of different purity grades of OVA with the optimized chemical mobilization method applying glutamic acid (pH 10.50). The sample contained 0.8% m/v PL 3–10, 2.0% m/v PL 5–6, 10.7 mmol/L L-Arg, 1.8 mmol/L IDA, and CIEF gel, with (a) 0.36 mg/mL OVA grade V; (b) 0.19 mg/mL OVA grade VI; (c) 0.44 mg/mL OVA grade II. Samples were focused for 15 min at +25.0 kV. Mobilization was done by replacing the catholyte with 50 mmol/L L-Glu (pH 10.50) and application of +30.0 kV.
performed in two-step CIEF. When comparing pressure and chemical mobilization modes, mobilization with an acidic zwitterion provided best selectivity. For either mobilization approach, optimization of the composition and individual concentration of CAs is essential in realizing improved resolution. However, when the employed narrow pH range CA covered the target pI of some model compounds and exceeded threshold concentration, peak duplication due to incomplete focusing might occur that could only be counteracted in part by increase in focusing time and voltage. Spacer compounds added to prevent CA losses caused by ITP-driven processes exert a favorable effect on stabilizing the pH gradient, but increased the baseline noise in the focusing region of OVA with pressure mobilization. Increasing the cathodic spacer concentration additionally dislocated alkaline constituent zones from behind the detector, but impaired resolution by compression of the pH gradient and increased background signals with pressure mobilization. Thus, spacer concentration can be tailored according to whether the entire pI gradient offered by CAs has to be addressed or improved selectivity in the acidic domain is required. In the latter case, the loss of alkaline and possibly also neutral analytes might occur. Application of the optimized two-step CIEF employing mobilization with an acidic zwitterion revealed differences in the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Theresa Kristl Hanno Stutz
Research Article
Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants†
Received September 1, 2014 Revised October 20, 2014 Accepted October 20, 2014
One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1–10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3–10 with Pharmalyte 5–6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5–6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. L-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. L-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed. Keywords: Capillary isoelectric focusing / Chemical mobilization / Food allergens / Ovalbumin / Pressure mobilization DOI 10.1002/jssc.201400890
1 Introduction The characterization of proteins, for example, by product profiling, constitutes an important aspect of quality assurance. In biological and biopharmaceutical products, for example, (recombinant) allergens, post-translational modifications (PTMs) but also process- and product-related impurities [1] increase the number of protein species and thus the product complexity. In this context, CE has been applied to separate closely related allergen variants [2–4]. Food allergens affect up to 8% of children and represent a key source of allergic disorders [5]. Ovalbumin (OVA), an albumin species most abundant in egg white [6], is a major causative elicitor of food allergies [7] encountered not only in eggs and related products, but also in processed food [8] where egg white is added to provide additional nutrients and improve flavor or texture [9]. Food with OVA contamination or without appropriate declaration are thus detrimental for allergic patients [10]. Moreover, OVA is also present in Correspondence: Dr. Hanno Stutz, Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria E-mail: [email protected] Fax: +43-662-8044-5751
Abbreviations: CA, carrier ampholyte; Ca II, carbonic anhydrase isoform II; IDA, iminodiacetic acid; OVA, ovalbumin; PL, Pharmalyte; PTM, post-translational modification C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
vaccines produced in chicken eggs and shows content fluctuations in seasonal vaccine batches [11]. In addition, OVA is used as a prominent allergen frequently applied for sensitization and challenging experiments in immunological studies [12, 13], for example, in asthma mouse models [14]. OVA is composed of 385 amino acids with 42.9 kDa, possesses one acetylation, two phosphorylation sites, and an N-glycosylation at Asn292 [15, 16]. Different occupations of acetylation and phosphorylation sites influence the pI. Recently, an additional PTM decoration, that is, by nitration, was shown to enhance the allergenicity of modified OVA [17]. Moreover, commercial OVA products were shown to contain copurified minor impurities [16]. The manifold applications of OVA emphasize the importance of an appropriate standardization and profiling of commercial products that are used in immunological studies. A cross-evaluation of applied OVA products proving equivalency or revealing differences is thus supportive for the comparability of immunological results. Within the arsenal of bioanalytical techniques for distinction of intact proteins and PTMs, CE has conquered a distinguished position due to its outstanding separation efficiency. Among its different modes, particularly CIEF is considered to provide outstanding selectivity [18–21]. The separation † This paper is included in the virtual special issue on Amino acids, proteins and peptides available at the Journal of Separation Science website.
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principle addresses differences of pI according to Eq. (1) for resolution in (C)IEF: D.[d(pH)/dx] ⌬(pI) = 3 (1) E .[−d/d(pH)] and theoretical differences down to ⌬0.02 pI units can be resolved [22]. Thereby, D represents the diffusion coefficient of the analyte, d(pH)/dx is the spatial change of pH within the generated gradient, −d/d(pH) is the mobility change with pH at the respective pI, and E is the electric field strength. The fundamentals of CIEF within a pH gradient formed by commercial carrier ampholytes (CAs) have been reviewed comprehensively [23–25]. The primary pH gradient generated by the applied anolyte and catholyte solutions is stabilized by CAs [26]. Since the quality of CAs varies [27, 28] and will influence the separation performance, an appropriate CA selection and blending different CAs that cover different pH domains have also proven their importance in establishing selectivity. This can be achieved by a local widening of the gradient within the pH domain that covers the pI of target analytes [3, 29]. However, composition of the CA mixture is delicate to prevent extended focusing caused by analyte retardation due to progressive loss in net charge when approaching its focusing site. This might result in incomplete focusing [22, 30]. Moreover, analyte focusing in the distal capillary part behind the detector, and time-dependent changes of the pH gradient by ITP-driven losses of highly acidic and alkaline CAs at the capillary ends are essential limitations [31,32]. Reagents that block the very acidic and alkaline sections, that is, so-called spacer, counteract these drawbacks [29–31, 33], but have to be adjusted to the respective separation challenge to fulfill their task without compromising the resolution by compressing the pH gradient via substantial occupation of the terminal capillary parts. Different mobilization strategies have been developed and applied in CIEF, including mobilization by pressure and chemical means [34]. Although advantages and disadvantages of the respective strategies have been discussed, a competitive comparison of mobilization approaches for the same analyte implementing an individual tuning of CAs is still neglected. Since OVA with its various PTMs and concomitant subtle differences in the pI can be considered a challenging analyte, differences in the potential of the respective mobilization strategies will become evident. This work targets to provide a comparative evaluation of mobilization strategies for two-step CIEF. From a common CIEF basis, individual mobilization strategies are set and optimized followed by a subsequent refinement of the CIEF focusing step, respectively. The most distinguished CIEF method will then be employed for profiling and comparison of various commercial OVA products.
2 Materials and methods 2.1 CE Measurements were performed on the Agilent 7100 Capillary Electrophoresis System (Agilent Technologies, Waldbronn, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Germany). The 7100 CE system was equipped with a diodearray detector covering 190–600 nm. For cutting-off low wavelengths that would interfere with absorbance of CAs, detector filter assembly G7100–62700 (from Agilent Technologies) was used for executing CIEF at 270 nm. Band width and scan rate were set to 4.0 nm and 2.5 Hz, respectively. Data treatment was done with Chem Station, Rev. B.04.03. For CIEF separation, eCAPTM neutral coated CIEF capillaries (Beckman CoulterTM , La Brea, CA, USA) with 50 m id, effective capillary length to detector (LD ) 23.0–23.9 cm, and total capillary length (LT ) 31.4–32.6 cm were aligned in the interface for 50 m straight capillaries. The cassette temperature was set to 20.0⬚C. The current limit was selected with 20 A according to the recommendation of the capillary manufacturer to protect the capillary coating.
2.2 Chemicals Sodium hydroxide (w = 32%), orthophosphoric acid (H3 PO4 , w = 85%), and acetic acid (glacial; 100%) were obtained from Merck (Darmstadt, Germany). L-Arginine (L-Arg) and iminodiacetic acid (IDA)—used as spacer solutions— and L-glutamic acid (L-Glu; for chemical mobilization) were from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was prepared by a Milli-Q Plus 185 system (Millipore, Molsheim, France). The cIEF gel was acquired from Beckman Coulter. CAs PharmalyteTM pH 3–10 and PharmalyteTM pH 5–6 were obtained from GE Healthcare (Piscataway, NJ, USA). Anolyte and catholyte stock solutions were 1.00 mol/L H3 PO4 and 1.00 mol/L NaOH, respectively. Stock solutions of cathodic and anodic spacer were 500 mmol/L L-Arg and 200 mmol/L IDA, respectively, as outlined elsewhere [33]. For chemical mobilization different stock solutions were prepared, including 2.20 mol/L acetic acid, and 50 mmol/L L-Glu. The pH of 50 mmol/L L-Glu was either adjusted to 10.50 with 1.00 mol/L NaOH for application with normal pH gradient (acidic domain at capillary inlet), or to 2.15 by adding 85% and 1.00 mol/L H3 PO4 when applied in the reversed pH gradient, respectively. All stock solutions were stored between +4 and +8⬚C and proved stability for at least 4–5 months under these conditions.
2.3 Proteins and pI markers Albumin from chicken egg white (OVA) was purchased from Sigma-Aldrich in different purities specified as grade II (crude dried egg white 62–88% OVA), grade V (ࣙ98% purity), and grade VI (ࣙ98% purity and low mannose content) by the manufacturer. Stock solutions of OVA were prepared in ultrapure water at 5.83 mg/mL (grade II), 16.75 mg/mL (grade V), and 2.52 mg/mL (grade VI). Solutions of OVA grade II and VI were filtered due to slight precipitates. Myoglobin from horse heart (min. 90%) and carbonic anhydrase isoform II (Ca II from bovine erythrocytes, pI 5.91 [35] and 5.4) were obtained from Sigma-Aldrich. Peptide markers with pI 10.0, 9.5, www.jss-journal.com
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7.0, 5.5, and 4.1 and primary sequence according to Shimura et al. [35], and ribonuclease A (pI 9.68 [35]) were obtained from Beckman Coulter. Synthetic peptides with pI 4.72 (P1) and pI 4.51 (P2) were synthesized on demand with a purity >95% by Metabion International AG (Martinsried, Germany). The pI values of these peptides have been tailored by the authors via selection of appropriate amino acid sequences to flank OVA variants as closely as possible. Stock solutions of horse heart myoglobin (1.00 mg/mL), ribonuclease A (24.00 mg/mL), Ca II (pI 5.4; 4.06 mg/mL), Ca II (pI 5.91; 4.00 mg/mL), P1 (0.95 mg/mL), and P2 (1.00 mg/mL) were prepared in ultrapure water. Aliquots of 25 L were prepared from stock solutions and stored at −20⬚C. After thawing, aliquots were rapidly consumed. CIEF samples contained 81.5% v/v cIEF gel, spacer compounds in appropriate concentrations, model peptides and proteins, and OVA. The sample composition varied and details are given in the respective sections and figure captions.
2.4.1.2 Chemical mobilization with acetic acid or glutamic acid (pH 10.50) CIEF was performed with 200 mmol/L H3 PO4 in cIEF gel (anolyte) and 300 mmol/L NaOH (catholyte) [33] applying +25.0 kV for 15.0 min. For chemical mobilization the catholyte was either replaced with (i) 22 mmol/L acetic acid or (ii) 50 mmol/L L-Glu (adjusted to pH 10.50) with application of +30.0 kV [36].
2.4 CIEF
3 Results and discussion
Pretreatment of eCAP neutral capillaries is outlined elsewhere [3]. Briefly, new eCAP capillaries were conditioned with 10 mmol/L H3 PO4 for 4 min followed by ultrapure water for 15 min (900 mbar each). Before the first daily CIEF separation, the capillary was flushed with ultrapure water for 15 min (900 mbar). Before each CIEF run, the eCAP capillary was flushed with 90.9 mmol/L H3 PO4 in cIEF gel for 2 min followed by ultrapure water for 4 min (both 900 mbar). Anolyte, catholyte, and sample solutions were prepared freshly and vortexed thoroughly followed by a centrifugation step with 18 600 × g for 5 min at 20⬚C to remove air bubbles and particulate matter before their use. Samples were injected hydrodynamically with 900 mbar for 200 s to assure filling of the entire capillary with the sample. Capillary ends were dipped into ultrapure water after injection to avoid carryover. Settings for subsequent focusing and the optimized mobilization steps are specified in Section 2.4.1. Anolyte and catholyte solutions were used for two to four runs depending on CIEF analysis time to prevent electrolyte depletion. After each CIEF run, the capillary was flushed with ultrapure water for 4 min (900 mbar). Daily measurements were finished by filling the eCAP capillary with cIEF gel and subsequent storage at +4 to +8⬚C with capillary ends dipped into ultrapure water.
3.1 Optimization of spacer concentrations
2.4.1 Focusing and mobilization 2.4.1.1 Pressure mobilization During CIEF focusing, +15.0 kV were applied for 6 min with 90.9 mmol/L H3 PO4 in cIEF gel as anolyte and 19.2 mmol/L NaOH (in water) as catholyte. During subsequent pressure mobilization 48 mbar were applied on the anodic site with auxiliary +21.0 kV. Focusing setting is slightly different from strategies using chemical mobilization due to baseline irregularities. Details are given in the text. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4.1.3 Chemical mobilization with reversed pH gradient and glutamic acid (pH 2.15) Positions of anolyte (200 mmol/L H3 PO4 in cIEF gel) and catholyte (300 mmol/L NaOH in water) [33] were changed thus reversing the pH gradient with the acidic domain situated at the detector side. Focusing was done at −25.0 kV for 15.0 min. For mobilization, the anolyte was exchanged against 50 mmol/L L-Glu (pH 2.15) with application of −30.0 kV.
The implementation of spacer compounds in the CIEF sample ensures temporal stabilization of the pH gradient by preventing CA losses to electrolyte vessels. This contributes to an enhanced repeatability of mobilization times [33, 37]. Efficient spacer compounds should ideally act as good CAs [23] and additionally occupy the pI gap between most acidic CAs and the anolyte and most alkaline CAs and the catholyte, respectively [33]. Therefore, L-Arg (pI 10.76) [22] and IDA (pI 2.23) [38] were selected as cathodic and anodic spacer compounds as described recently [32,33]. Concentrations and thus zone lengths of spacer can be selected individually since IDA has to overcome anodic losses of CAs, whereas L-Arg prevents cathodic CA losses and may additionally block the distal capillary part behind the detector according to requirements. 3.1.1 Spacer concentrations with pressure mobilization Different spacer concentrations were tested with pressure mobilization to reveal their effects on selectivity and detection, applying a mixture of model proteins/peptides and a preoptimized CA mixture of 0.8% m/v Pharmalyte 3–10 (PL 3–10) and 0.5% m/v Pharmalyte 5–6 (PL 5–6; Fig. 1). L-Arg was tested at 0, 10.7, 21.4, and 42.9 mmol/L with highest concentrations referring to a previous test series [33] in combination with 0.4–1.4 mmol/L IDA. Without spacer addition, intensities of peaks presumed to represent alkaline and neutral analytes were too low for an unambiguous assignment (data not shown). Increased L-Arg concentrations progressively enhanced signal intensities primarily in the alkaline domain that also explains visibility of so-called cathodic peaks at higher L-Arg concentrations (Fig. 1, traces a–c). Both effects www.jss-journal.com
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Figure 1. Influence of spacer concentrations on the CIEF separation with pressure mobilization. The sample contained 0.8% m/v PL 3–10, 0.5% m/v PL 5–6, 0.43 mg/mL ribonuclease A (RnA), 0.038 mg/mL horse heart myoglobin (HHM), 0.036 mg/mL Ca II (5.91), 0.045 mg/mL Ca II (5.4), 10.15 g/mL P1, 5.35 g/mL P2, and cIEF gel, including the following spacer combinations: (a) 10.7 mmol/L L-Arg + 0.4 mmol/L IDA, (b) 21.4 mmol/L L-Arg + 0.9 mmol/L IDA, (c) 42.9 mmol/L L-Arg + 1.4 mmol/L IDA. Anolyte: 90.9 mmol/L H3 PO4 in cIEF gel; catholyte: 19.2 mmol/L NaOH. Either sample was focused for 6 min at +15.0 kV. Mobilization was done by 48 mbar at +21.0 kV. Arrows indicate region of baseline irregularities.
can be related to a progressive compression of the pH gradient by the spacer. Cathodic peaks occur due to a transient bidirectional head-to-head movement of analytes from either capillary end toward their focusing position [30, 31, 33]. In the alkaline to neutral pH region, the resolution improved, whereas it decreased in the acidic region when L-Arg concentration was raised. This discrepancy as well as the prominent increase in peak heights in the region between pH 6 and 10 might be related to a nonuniform compression of the pH gradient, whereby the acidic part that additionally contains PL 5–6 is less affected. However, currently it is not clear without ambiguity whether alkaline and neutral peaks at 10.7 mmol/L L-Arg represent focused zones or anodic peaks on their way toward the final focusing site behind the detector that are prematurely mobilized (Fig. 1, trace a). The increase to 21.4 and 42.9 mmol/L L-Arg would shift the focusing position in front of the detector (compare results of Section 3.1.2). This would offer an alternative explanation for improved resolution and increased peak heights (Fig. 1, traces a–c), since compression of the pH gradient would accelerate focusing, assure its completion, and result in increased peak heights. In parallel, the combined focusing and mobilization time (tm ) of model proteins and peptides increased due to the accretive occupation of the distal capillary part by L-Arg and the concomitant increase in distance that analyte zones are focused ahead of the detector. Remarkably, baseline irregularities occurred within the focusing region of OVA (pI 4.51–4.72) when increasing L-Arg (Fig. 1, traces a–c). These baseline irregularities are apparently related to the applied wide pH range CA, that is, PL 3–10, in interplay with L-Arg concentration. Concentration reduction of both PL 3–10 and L-Arg attenuated baseline irregularities. The described effects were duplicated with different batches of PL 3–10 and also by replacing L-Arg with other cathodic spacers, that is, L-Lys or N,N,N ,N -tetramethylethane1,2-diamine (TEMED) [22, 39] (data not shown). Moreover, an increase in the focusing time, for example, to 15 min, also increased background fluctuations. Evidently, a slightly unequal distribution of CA species within this pH regime
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together with the compression of pH gradient could yield the baseline unsteadiness. No comparable effects were encountered for IDA up to the highest concentration tested, that is, 10 mmol/L (data not shown). Irrespective of the proposed explanatory model, low L-Arg concentrations would favor improved resolution in the acidic domain and thus of OVA and reduce the UV background at the expense to miss alkaline constituents. 3.1.2 Chemical mobilization The effects of spacer and CA described in the previous section could not be transmitted a priori to chemical mobilization with acetic acid. Thus, testing was repeated but confined to L-Arg concentrations of 10.7, 21.4, 42.9 mmol/L, maintaining the IDA concentration at 1.8 mmol/L, respectively. Contrary to the pressure mobilization, only 1.8% m/v PL 3–10 without PL 5–6 was applied. The increase in peak heights of alkaline to neutral marker was not observed when rising L-Arg. This seems to corroborate the derived explanatory model considering also that PL 5–6 was not included. However, L-Arg had to exceed 20 mmol/L to allow for the detection of marker compounds with pI 10.0 and 9.5 by sufficient occupation of the capillary blind end as corroborated by recent results [33]. Although the cathodic spacer slightly increased the baseline noise, pronounced irregularities as in pressure mobilization were absent except for a small rectangular step at pI < 4.1 that will not interfere with OVA (data not shown).
3.2 Optimization of CA composition for different mobilization strategies 3.2.1 Pressure mobilization After the selection of spacer concentrations, different combinations and concentrations of wide and narrow pH range
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J. Sep. Sci. 2015, 38, 148–156 Figure 2. Optimization of CIEF with pressure mobilization for OVA (grade V) by various CA combinations all given as m/v. (A) Resolution improvement by continuous reduction of PL 5–6 maintaining 0.8% PL 3–10 throughout all CIEF experiments. Traces: (a) 1.5% PL 5–6, (b) 1.0% PL 5–6, (c) 0.3% PL 5–6. (B) Resolution improvement of OVA fractions by variation of PL 3–10 maintaining 0.5% PL 5–6 throughout all CIEF experiments. Traces: (a) 1.5% PL 3–10, (b) 0.8% PL 3–10, (c) 0.3% PL 3–10. (C) CIEF separation with optimized composition of wide and narrow range CAs. Sample: 0.3% PL 3–10, 1.0% PL 5–6, 0.36 mg/mL OVA (grade V) with (a) 10.7 mmol/L L-Arg and 1.8 mmol/L IDA, (b) no spacer added. Signal fractions of OVA are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
CAs were tested to optimize the pH gradient in the focusing region of OVA. Based on an initial preoptimization, a combination of 0.8% m/v PL 3–10 and 0.5% m/v PL 5–6 (see Section 3.1.1) was selected as starting point. During the optimization procedure, one CA was maintained and the other varied. When PL 3–10 was kept at 0.8% m/v, PL 5–6 was varied in six incremental steps from 0.3 to 2.0% m/v, selecting 10.7 mmol/L L-Arg and 1.8 mmol/L IDA as spacer. L-Arg was slightly reduced to lower baseline signals and improve resolution since OVA is focused on the capillary inlet part. Best resolution of OVA was achieved with 0.8% m/v PL 3–10 and 1.0% m/v PL 5–6 (Fig. 2A) with three major peaks (peaks 2–4), a minor peak (peak 6), and an additional shoulder at the most acidic major OVA fraction (peak 5). Lower and higher PL 5–6 concentrations progressively deteriorated resolution (Fig. 2A, traces a–c). Subsequent optimization addressed the variation of PL 3–10 between 0.3 and 1.8% m/v, keeping the original PL 5–6 content, that is, 0.5% m/v, revealing best resolution at 0.3–0.5% m/v PL 3–10 (Fig. 2B, traces a–c). The low PL 3–10 concentration favorably reduces the baseline irregularities in the OVA domain. A final optimization selecting 0.3% m/v PL 3–10 with further testing of PL 5–6 closely around the previously optimized 0.8% revealed a slightly higher PL 5–6 content more advantageous. Thus, the finally optimized CA combination comprised 0.3% PL 3–10 with 1.0% (all m/v) PL 5–6 in combination with 10.7 mmol/L L-Arg and 1.8 mmol/L IDA (Fig. 2C, trace a). When OVA was analyzed with this CA combination, but in absence of spacer, resolution was substantially improved (Fig. 2C, trace b). This is due to an expansion of the pH gradient and the reduction of the preponderant contribution of the described baseline anomaly that interferes with OVA. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2.2 Chemical mobilization with acetic acid For chemical mobilization with a weak acid, that is, acetic acid, initial testing was confined to PL 3–10 with 1.8% m/v. Mobilization was done by replacing the catholyte with acetic acid during the mobilization step. Concentrations between 91.0 and 200 mmol/L H3 PO4 (anolyte) and between 18.9 and 300 mmol/L NaOH (catholyte), respectively, showed no substantial difference in resolution and peak shapes of model peptides (pI 4.1–10.0). Therefore, the highest tested anolyte and catholyte concentrations were kept for repeatability reasons [33]. Moreover, a stepwise increase of the acetic acid concentration in the mobilizer solution from 22 to 175 mmol/L showed no difference in the separation result and thus 22 mmol/L was selected. All further CIEF measurements were performed with spacer concentrations optimized for pressure mobilization (see Section 3.2.1), that is, 10.7 mmol/L L-Arg, 1.8 mmol/L IDA. CA optimization was started by stepwise increase of PL 3–10 from 1.2 to 2.8% m/v (without PL 5–6; data not shown). The best PL 3–10 concentration, that is, 1.8%, was kept and then incrementally supplemented with 0.3–2.5% m/v PL 5–6. Resolution of OVA fractions improved up to 1.5% PL 5–6 and remained virtually unaffected by higher PL 5–6 concentrations (Fig. 3A, traces a–c). This is related to a pronounced capillary occupation of pH 5–6 section and adjacent parts within the pH gradient, which causes disappearance of peptide pI 7.0 starting with 2.0% m/v PL 5–6 due to its focusing behind the detector. With 1.5% m/v PL 5–6, this peptide occurred again within the focusing step. In parallel, peptide pI 5.5 started to split in two separate peaks at 0.8% m/v PL 5–6, with increasing spatial distance between these twin peaks at higher PL 5–6 concentrations (data not www.jss-journal.com
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Figure 3. Optimization of CAs (all given as m/v) for chemical mobilization with 22 mmol/L acetic acid. Details of CIEF separation depicted for the focusing domain of OVA (grade V). (A) Resolution improvement of OVA fractions by variation of PL 5–6 with 1.8% PL 3–10 throughout all CIEF experiments. Traces: (a) 0.3% PL 5–6, (b) 1.5% PL 5–6, (c) 2.5% PL 5–6. (B) Resolution improvement by variation of PL 3–10 maintaining 1.5% PL 5–6. Traces: (a) 0.5% PL 3–10, (b) 0.8% PL 3–10, (c) 2.8% PL 3–10. Separated fractions are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
shown). In a second approach the optimized content of 1.5% m/v PL was kept with variation of PL 3–10 between 0.5 and 2.8% m/v. The combination of 0.8% PL 3–10 and 1.5% m/v PL 5–6 provided best resolution (Fig. 3B, traces a–c) with separation pattern nearly congruent to 1.8% PL 3–10 and 1.5% m/v PL 5–6, but at faster tm (Fig. 3A and B, traces b, respectively).
3.2.2.1 Signal splitting The described peak splitting of peptide pI 5.5 (see Section 3.2.2) has been related to insufficient focusing under the selected conditions. Increasing the relative PL 5–6 content from 0.8 to 2.5% in the CA mixture, keeping 1.8% (all m/v) PL 3–10 progressively enhanced the time interval between pI 5.5 twin peaks, whereas an increase in the portion of PL 3–10 (from 0.5 to 1.8% m/v with constant PL 5–6) reduced their time difference without achieving complete mergence. Sole operation with PL 3–10 ceased the splitting effect (data not shown). This phenomenon can be explained by the continuous retardation in velocity during the focusing step due to a concomitant reduction in net charge when approaching the focusing region [22]. For synthetic peptides (pI 4.72 and 4.51) flanking OVA, these effects were absent thus ruling out contribution of artificial peak splitting to the observed OVA heterogeneity (data not shown). This justifies also the application of a narrow pH range CA with a slight offset from the pI of OVA. Since CA species extent over their nominal range [3, 40], their leakage in the pI region of interest will improve the resolution by an appropriate flattening of the pH slope without requiring extended C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
focusing times that inherently also bear the risk of protein precipitation. 3.2.3 Chemical mobilization with zwitterion Alternatively, a chemical mobilization with a zwitterion, that is, 50 mmol/L L-Glu, was tested according to ref. [36]. Therefore, the pI of the mobilizing zwitterion (L-Glu pI = 3.22) [41] was selected below that of OVA to assure focusing of L-Glu behind the analyte and thus a mobilization of the analyte toward the detector. The spacer concentrations and initially also the CA combination that had been optimized for acetic acid were both maintained. Alike for acetic acid, baseline noise in the focusing region showed only a tiny increase compared to the overall baseline. Again, the CA composition was refined varying PL 3–10 between 0.5 and 1.2%, while maintaining 1.5% (all m/v) PL 5–6 (Fig. 4A, traces a–c). Subsequently, PL 5–6 was varied between 1.0 and 2.0% (Fig. 4B, traces a–c) with 0.8% (all m/v) PL 3–10 retained as most favorable as evident from the previous optimization (Fig. 4A, trace b). The combination of 0.8% PL 3–10 and 2.0% m/v PL 5–6 provided best selectivity with resolution of two major and ten minor peaks (Fig. 4B, trace c). 3.2.4 Chemical mobilization with zwitterion and reversed pH gradient Due to a total CIEF analysis time (i.e., focusing and mobilization) of approximately 33 min (Fig. 4B, trace c), acceleration was aimed by a reversal of anolyte and catholyte, and thus the pH gradient (acidic part at capillary outlet; see www.jss-journal.com
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Figure 4. Resolution improvement of OVA (grade V) via optimization of CA composition (all given as m/v) for chemical mobilization with 50 mmol/L L-Glu (pH 10.50). (A) Variation of PL 3–10 maintaining 1.5% PL 5–6. Traces: (a) 0.5% PL 3– 10, (b) 0.8% PL 3–10, (c) 1.2% PL 3–10. (B) Variation of PL 5–6 maintaining 0.8% PL 3–10. Traces: (a) 1.0% PL 5–6, (b) 1.5% PL 5–6, (c) 2.0% PL 5–6. Separated fractions are numbered chronologically in the order of their occurrence. The baseline irregularities (stemming from the background) are annotated with asterisks.
Section 2.4.1.3). The idea is to focus OVA intentionally behind the detector in the blind part of the capillary requiring subsequent mobilization toward the capillary inlet with OVA zones approaching the detector from its rear. This will substantially reduce the required mobilization time. The CA combination from Section 3.2.3 was maintained, including only IDA, but not L-Arg. Indeed, the mobilization time was considerably accelerated but some minor fractions were already detected during focusing. In total, four fractions were distinguished. When the focusing time was increased from 10 to 20 min to exclude presence of artifact peaks, the peak pattern remained unchanged excluding insufficient focusing (data not shown). To avoid focusing within the detection window, L-Arg was incrementally added (0–36.6 mmol/L), whereas IDA was omitted. With 36.6 mmol/L L-Arg, OVA was entirely pushed to the blind capillary end and CIEF analysis time was 24 min (Fig. 5A and B). However, selectivity was impaired and similarly to pressure mobilization baseline irregularities that interfered with some OVA fractions were introduced.
3.3 Profiling of commercial OVA products Based on the results of the optimized CIEF mobilization strategies, chemical mobilization with 50 mmol/L L-Glu pH 10.50 was selected most appropriate and applied for profiling different commercial OVA products, specified as grade II, V, and VI (Fig. 6, traces a–c). Grade V and VI showed only marginal differences as expected from the specifications of the manufacturers (see Section 2.3). Two major fractions (peaks 5 and 8, Fig. 6) and 8–10 minor fractions were separated (Fig. 6). The relative abundance of the major peaks C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. CIEF optimization for chemical mobilization with 50 mmol/L L-Glu (pH 2.15) with reversed pH gradient. (A) Application of different L-Arg concentrations: (a) no L-Arg, (b) 10.7 mmol/L L-Arg, (c) 21.4 mmol/L L-Arg, (d) 36.6 mmol/L L-Arg. The sample contained 0.8% m/v PL 3–10, 2.0% m/v PL 5–6, 0.36 mg/mL OVA (grade V), 5.35 g/mL P2 (pI 4.51) and cIEF gel. The sample was focused for 15 min at −25.0 kV. Mobilization was done by replacing the anolyte with 50 mmol/L L-Glu (pH 2.15) and application of −30.0 kV. (B) Detail of the optimized CIEF separation pattern with 36.6 mmol/L L-Arg.
was nearly congruent for both products. In case of grade II, the alkaline major fraction (peak 5) was considerably reduced, whereas acidic variants (peaks 9 and 10) were more prominent (Fig. 6, trace a).
4 Concluding remarks A comprehensive comparison of different mobilization strategies for a major food allergen, that is, OVA, has been www.jss-journal.com
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constituent profile of three commercial OVA products and proved the suitability of the method for the outlined aim. Agilent Technologies (Waldbronn, Germany) is kindly acknowledged for providing an Agilent 7100 CE System as a loan in the course of a Research Sponsoring Agreement. The authors are indebted to Dr. Gerard Rozing, Dr. Martin Greiner, and Dr. Hans-Josef Brunnert (all from Agilent Technologies, Waldbronn) for their support within the research cooperation. The authors have declared no conflict of interest.
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Figure 6. Testing of different purity grades of OVA with the optimized chemical mobilization method applying glutamic acid (pH 10.50). The sample contained 0.8% m/v PL 3–10, 2.0% m/v PL 5–6, 10.7 mmol/L L-Arg, 1.8 mmol/L IDA, and CIEF gel, with (a) 0.36 mg/mL OVA grade V; (b) 0.19 mg/mL OVA grade VI; (c) 0.44 mg/mL OVA grade II. Samples were focused for 15 min at +25.0 kV. Mobilization was done by replacing the catholyte with 50 mmol/L L-Glu (pH 10.50) and application of +30.0 kV.
performed in two-step CIEF. When comparing pressure and chemical mobilization modes, mobilization with an acidic zwitterion provided best selectivity. For either mobilization approach, optimization of the composition and individual concentration of CAs is essential in realizing improved resolution. However, when the employed narrow pH range CA covered the target pI of some model compounds and exceeded threshold concentration, peak duplication due to incomplete focusing might occur that could only be counteracted in part by increase in focusing time and voltage. Spacer compounds added to prevent CA losses caused by ITP-driven processes exert a favorable effect on stabilizing the pH gradient, but increased the baseline noise in the focusing region of OVA with pressure mobilization. Increasing the cathodic spacer concentration additionally dislocated alkaline constituent zones from behind the detector, but impaired resolution by compression of the pH gradient and increased background signals with pressure mobilization. Thus, spacer concentration can be tailored according to whether the entire pI gradient offered by CAs has to be addressed or improved selectivity in the acidic domain is required. In the latter case, the loss of alkaline and possibly also neutral analytes might occur. Application of the optimized two-step CIEF employing mobilization with an acidic zwitterion revealed differences in the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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