Separation of Polypeptides by Isoelectric Point ... - MS Wil GmbH

Jul 9, 2012 - ABSTRACT: We introduce an online multiple-junction capillary isoelectric focusing fractionator .... prior to MS detection via online RPL...
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Separation of Polypeptides by Isoelectric Point Focusing in Electrospray-Friendly Solution Using a Multiple-Junction Capillary Fractionator Konstantin Chingin,† Juan Astorga-Wells,†,‡ Mohammad Pirmoradian Najafabadi,†,‡ Thorleif Lavold,‡ and Roman A. Zubarev*,†,§ †

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden Biomotif AB, Stockholm, Sweden § Science for Life Laboratory, Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.

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separation, excellent concentration capacity, high resolving power, and sensitivity of CIEF have been found particularly useful in the analysis of protein mixtures from biological samples.3−6 In combination with electrospray ionization (ESI)MS detection, CIEF has been applied in a number of protein studies for identification purposes7−13 as well as to probe protein refolding,14 phosphorylation,15 glycosylation,16 noncovalent interactions,17 or to determine the isoelectric point of protein isoforms.18 Because CIEF alone is often insufficient for comprehensive analysis of real biological samples, the attention is gradually shifting toward employing this method in multidimensional platforms as a preparative step for a second-dimension separation.4 Of particular interest is the hyphenation between CIEF and RPLC, which offers a promising alternative to the off-line 2D-PAGE approach19 in high-throughput proteomics analyses. Unlike 2D LC × LC protocols, the retention principles of CIEF and RPLC are truly orthogonal, which greatly benefits the overall peak capacity in this type of hyphenation.20,21 A number of CIEF−RPLC−MS

remendous developments in mass spectrometry (MS) over the past 2 decades have brought it to the forefront of bioanalytical methods. Unequaled specificity, resolving power, mass accuracy, and high-throughput capabilities offered by modern MS instrumentation render it essentially indispensable in many types of biomolecular studies, particularly in the fields of proteomics and metabolomics.1,2 The practicality of complementary bioanalytical methods is therefore becoming increasingly dependent on their online compatibility with MS analysis rather than their mere stand-alone performance. This trend is nicely illustrated by the current prevalence of liquid chromatography (LC), in particular reversed-phase LC (RPLC), over other separation techniques. While several other types of separations techniques, such as isoelectric focusing (IEF), capillary zone electrophoresis (CZE), or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE), are capable of providing superior operation in a number of biological applications, it is the poor efficiency of online interfaces with MS detection that limits the wider application of these methods. Capillary isoelectric focusing (CIEF) is an electrophoretic method that separates amphoteric molecules in solution according to their isoelectric point (pI). Mild conditions of © 2012 American Chemical Society

Received: May 29, 2012 Accepted: July 9, 2012 Published: July 9, 2012 6856

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Figure 1. Schematic layout of the OMJ-CIEF setup: the six-port injector is shown in the sample-loop loading position. For details on assembling the fractionator and experimental procedure refer to the Supporting Information.

anticonvective media are either avoided in MS hyphenations or replaced by less detrimental chemicals, such as glycerol−water mixtures.13,18,32 Large amounts of carrier ampholytes (CAs) in CIEF greatly affect the sensitivity of MS detection, suppressing ionization of analyte molecules and introducing abundant spectral interferences.29,34,41,42 Although the presence of ampholytes can be tolerated in some applications, it has a particularly devastating effect in the analyses of real-life biological mixtures, hindering observation of trace analytes. Another problem associated with using high concentrations of CAs, which is often disregarded in the literature, is their impact on the performance of a mass spectrometer. Long-term exposure to the high concentrations of CAs (on the order of millimolar) and other low-volatility compounds, such as glycerol, inevitably results in accumulation of these substances in transfer lines and ion optics of the instrument, gradually degrading its performance. This affects data reproducibility and requires frequent workflow interruption for technical maintenance. Carrier ampholytes can be quite efficiently removed prior to MS detection via online RPLC separation24,28,34,39,43 or by microdialysis systems in protein analyses.26,35−37,44 In the absence of purification steps, sensitivity of MS detection can be improved by employing lower concentrations of CAs compared to standard CIEF protocols. However, this approach is commonly associated with decreased buffering capacity and smoothness of pH gradient, as well as peak broadening and resolution losses.4,41 Finally, MS-incompatible hydrophilic polymers, such as polyacrylamide and poly(vinyl alcohol), which are frequently used as capillary coatings to prevent electroosmotic flow during separation,14,15,29,34 tend to emanate into the liquid phase, e.g., upon local heating, and hinder the analysis.45−49 In this report, we introduce an online multiple-junction capillary isoelectric focusing (OMJ-CIEF) fractionator for tandem analyses of biomolecules in solution. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions (Figure 1). Each junction is communicated with its own external electrolytic buffer, which is used both to supply electrical contact and for solvent exchange. The multiplejunction design adds a number of capabilities to the CIEF methodology. The possibility of local buffer exchange can be used to tailor the pH profile along the entire capillary, which makes it ideologically akin to the compartmental immobilized pH gradient (IPG) IEF fractionators. Furthermore, by applying a voltage gradient across certain segments of the capillary after the focusing stage, analyte fractions can be selectively mobilized for further interrogation by orthogonal methods. Control over the mobilization of focused fractions is of particular value for

platforms have been developed and applied in analyses of proteins and peptides from tumor tissues,22 human saliva,23 yeasts,24,25 and bacteria extracts.26−28 In spite of its unambiguous advantages for protein analysis, CIEF still remains in the shadow of other capillary separation methods in terms of routine utilization. A number of issues associated with online CIEF-MS sampling are still to be overpassed before this method can rival analytical characteristics and operability of mainstream protocols. The major constraints are briefly overviewed below. Unlike off-line analyses, the inherent absence of a terminal vial with electrolyte buffer in online CIEF-ESI-MS poses considerable technical challenge to ensure electrical continuity during the experiment. Most popular approaches to circumvent this problem employ coaxial sheath-liquid flow, which provides the necessary electrical contact on the capillary terminus during the focusing stage and aids subsequent ESI-MS detection.7,9,12,15,29−33 Simply speaking, the ESI source in such schemes is adapted to replace the outlet vial in classic CIEF experiments. This allows automated online operation but precludes the possibility of multidimensional analyses, such as CIEF−RPLC−MS. Furthermore, the use of sheath liquid causes analyte dilution at the tip of the capillary, decreasing the sensitivity of detection. Various microdialysis interfaces have been employed to supply catholytic and anolytic buffers with electric contacts in multidimensional CIEF setups,26,27,34−37 at the same time alleviating online purification of analyte fractions prior to MS detection.38 The high molecular weight cutoff in microdialysis membranes (∼3000 Da) makes them particularly suitable for protein applications but practically unfit for the analyses of peptides and other small molecules. Comprehensive online sampling of CIEF with the next-dimension separation in multidimensional approaches, e.g., with RPLC, represents another serious challenge. Normally, focused analytes are mobilized into different storage loops via a splitting interface, followed by a sequential RPLC−MS analysis of each loop.22,24,25,27,28,34,39 The efficiency of such sampling, however, suffers from the decreased resolution of separation during the mobilization step, especially when hydrodynamic flow is used.10,40 Besides that, a rapid valving system and the large number of storage loops are necessary to efficiently fractionate close CIEF bands. These and other technical requirements, e.g., the need for auxiliary pumping, sizably complicate experimental setup and operation. On the chemistry side, CIEF-MS analyses suffer from the low tolerance of MS toward classical CIEF reagents. Thus, inorganic acids and bases traditionally used in CIEF as catholytes and anolytes are normally replaced by ESIcompatible analogues, such as ammonia and formic acid.32,33 For the same reason, aqueous gels employed in CIEF as 6857

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multidimensional analyses that rely on CIEF separation as a first dimension. The fractionator is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms, such as ESI-MS or RPLC. We believe that OMJ-CIEF has the potential to become a practical add-on unit in a wide range of biomolecular studies, e.g., as a first-dimension separation in MS proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration. Here, we report on our proof-of-principle experiments in which the OMJ-CIEF fractionator was used online with ESI-MS detection for protein and peptide analyses.

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METHODS Details on assembling of the OMJ-CIEF fractionator, CIEF-MS experiments, protein digestion protocol, and chemicals and materials used in this study can be found in online Supporting Information.

Figure 2. Extracted ion chromatogram (EIC) for myoglobin as a function of focusing time in ammonium formate buffer (5 mM). Five CIEF-MS runs were conducted successively with different IEF times (0, 10, 35, 80, 180 min) using the same myoglobin stock solution for injection (10 μM in 5 mM ammonium formate). All the other experimental parameters were maintained unchanged in all the five runs. Separation was carried in the constant current regime (5 μA). EIC profiles are normalized to the peak intensity of the 80 min separation run.

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RESULTS AND DISCUSSION OMJ-CIEF in Ionic Buffers. The multiple-junction interface of OMJ-CIEF enables flexible external manipulation of the solution-phase composition across the separation capillary. In our first experiment, proteins were loaded in aqueous ammonium formate buffer, chosen for its good compatibility with MS.50 The same buffer was used as external electrolytic solution in all the Eppendorf vials. External solutions were titrated with ammonium hydroxide and formic acid such as to produce a linear pH gradient rising from 2.9 in the most downstream vial (no. 1 in Figure 1, MS end) up to 8.5 in the most upstream vial (no. 8 in Figure 1, injector end). The pH difference between each pair of adjacent vials was therefore equal to 0.8 units. The intention was to enforce a gradient inside the capillary during separation via local exchange of the liquid inside the capillary with external buffer solutions. This approach is conceptually different from the common IEF methodology of using internally added chemicals, such as ampholytes. To enhance the exchange rate, molarity of the outer buffer was 10-fold of the carrier solution (typically, 50 vs 5 mM). Figure 2 shows how focusing of myoglobin is achieved in time. It involves two major constituent processes: migration of the protein plug across the capillary and band narrowing. One can see that the axial displacement of the maximum of protein concentration is accomplished relatively fast while band narrowing takes typically twice as long (Supporting Information Figure S2). Subject to the external electric force, myoglobin eventually migrated to the downstream area of the capillary. From the elution peak time in MS, we estimate the focused myoglobin band to be located in between the second and the third junction of the capillary (Figure 1). In the focus point (zero net force), the pH value of the mobile phase should be equal to the pI of the protein, which is ca. 7.3 for myoglobin. Subsequently, pH should be higher than 7.3 upstream from the focus point and lower downstream in order to supply myoglobin with overall negative or positive charges, respectively. This conclusion is supported by simple pH-sensing experiments, in which the OMJ-CIEF was disconnected from MS after the focusing stage, and the effluent solution was continuously eluting onto a pH-indicator strip placed next to the downstream capillary terminal. A sigmoid profile was observed, started at pH ≈ 3−4 followed by a gradual increase up to pH ≈ 5−6 within first 3 min, and a subsequent abrupt

jump to pH > 10. Of course, this off-line sensing yields only a rough estimate, because high-mobility electrolytes rapidly diffuse inside the capillary as soon as the high voltage is switched off. Therefore, the actual slope of the sigmoid curve can be much steeper than measured. The inability to enforce a linear gradient using external buffers in our experiments can be explained by the high electrophoretic mobility of ammonium and formate ions. In the external electrical field abundant formate anions tend to migrate toward the positive pole, acidifying the downstream area of the capillary. In the same way, ammonium cations drift to the negative pole, resulting in the observed pH splitting. Kinetics of these processes appears to be faster compared to the solvent exchange rate through the Nafion membrane. This conclusion is supported by the reference experiments, in which a similar sigmoid profile was observed when all the external vials were filled with ammonium formate at pH 4. Formation of the upstream zone with pH > 10 in such experiments can only be explained by electrolyte redistribution along the capillary under applied voltage. A smoother gradient is produced when the two-component ammonium formate buffer is replaced with single-component ammonium hydroxide. The ratio between the concentrations of NH4+ and NH3 in 5 mM ammonium hydroxide solution is considerably smaller compared to that in neutral ammonium formate, when [NH4+] ≫ [NH3]. The low concentration of free NH4+ in the ammonium hydroxide solution prevents substantial redistribution of electrolytes when the external electrical field is applied. As a result, a low-sloped pH gradient from ca. 9 to 11 is established along the entire separation capillary. Figure 3 shows the separation of protein mixture containing myoglobin (pI 7.3), cytochrome c (pI 10.3), and lysozyme (pI 11.3) achieved in ammonium hydroxide. Being negatively charged in the entire pH range, myoglobin migrates downstream without constraints until it reaches the end of the column. It is worth noting that, in ammonium formate, myoglobin migration had been blocked higher up by the acidic pH zone rich with formate species, and therefore its elution took longer time (Figures 2 and 3). In contrast with myoglobin, lysozyme is positively charged in ammonium buffer and therefore drifts to the upstream end of the column. Finally, 6858

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the polarity of the IEF voltage was inverted by swapping over positive and negative electrodes, resulting in a mirror-image separation. A sharp peak of lysozyme was first detected in the ion chromatogram, followed by cytochrome c and, ultimately, a broad band of myoglobin. Besides the body of the fractionator, other factors contributing to peak distortion are associated with analyte transition through the T-unit, downstream Nafion membrane (no. 1, Figure 1), and PEEK tubing and fittings used to connect the fractionator with the ionization source. As a result, even the analytes focused at the downstream end of the OMJ-CIEF fractionator undergo notable broadening before detection. Peak broadening during analyte mobilization deteriorates the achievable separation and represents a general problem in CIEF. It can be partially alleviated by the use of capillaries with special coatings, anticonvective solution-phase additives, electroosmotic flow (EOF), or electrophoretic mobilization, etc., but these solutions are often associated with various adverse effects to the overall performance. Selective Analyte Mobilization in OMJ-CIEF. Complementary to mobilization protocols conventionally employed in CIEF, the multiple-junction design of OMJ-CIEF offers the capability to selectively release focused fractions. In this approach, bands focused downstream are mobilized to the detector with the carrier flow, while the upstream fractions are restrained by the electric voltage applied across the respective junctions. For example, in order to selectively release myoglobin in the CIEF experiment described above (Figure 3), the downstream electrode was transferred from the first to the third electrolyte vial and the carrier flow was then started. This way, myoglobin focused at the downstream end of the capillary freely eluted to the MS, while the release of cytochrome c and lysozyme was delayed by the trapping electric force. This electric force emerges as soon as the proteins start to migrate downstream and acquire positive charge in the lower pH region of the capillary. The force is increasing as the proteins drift further along the capillary and at some point can become sufficiently high to counterbalance the hydrodynamic force. The concept of trapping charged molecular species in membrane capillary interfaces by applied voltage is known as microfluidic electrocapturing51 and is employed in various bioanalytical applications for analyte purification,52,53 concentration,54,55 and separation56,57 purposes. Electrocapturing capacity depends on several experimental parameters, such as electrophoretic mobility of analyte ions at given pH, voltage magnitude, and hydrodynamic flow rate. In our experiments, we added 0.1% aqueous solution of formic acid in the first two vials right before the release. As a result, electrophoretic mobility of analyzed proteins sharply increased as they had reached the acidified downstream region of the fractionator. Under these conditions, capturing could be achieved at the carrier flow rate as high as 3 μL min−1, which enables rapid release of myoglobin (Supporting Information Figure S3). An alternative approach to enhance the efficiency of electrocapturing would be to use higher voltage, but it can be associated with various unwanted effects caused by Joule heating of the running solution. The selective analyte mobilization mode has several important advantages. As long as different bands are allocated by IEF into different capillary sections, their elution can be separated in time. This circumvents the problem of peak broadening and possible band overlapping during simultaneous mobilization, thereby increasing effective resolution of the

Figure 3. Separation of protein mixture containing myoglobin (10 μM), cytochrome c (65 μM), and lysozyme (56 μM) in ammonium hydroxide buffer (5 mM). IEF was carried over 80 min in the constant current regime (5 μA). All the fractions were mobilized for ESI-MS detection simultaneously via hydrodynamic pumping (3 μL min−1). EIC profiles of the proteins are normalized to the peak intensity of the myoglobin chromatogram.

cytochrome c is focused in the middle part of the capillary where the pH of the solution is equal to its pI value. To perform CIEF separation in a different pH range, ammonium hydroxide can be replaced with other ionic buffers compatible with MS detection. For example, aqueous acetic acid can be employed to generate a pH gradient in acidic range (pH ∼ 4−5). The major problem associated with this approach is that only narrow-range gradients can be produced and their profiles are highly dependent on electrolyte concentration. Thus, in the experiments described above the focus point of cytochrome c across the separation capillary was found to be particularly sensitive to the concentration of running buffer solution. Increasing molarity of ammonia would result in both higher mean value and narrower distribution of pH in the mobile phase due to the decreased [NH4+]/[NH3] ratio. This problem is obviated in CIEF when using zwitterionic ampholyte buffers. In the external electric field applied across the CIEF capillary, CAs will line up to locally modify the pH of the solution phase in accordance with their pI, resulting in a smooth and stable pH gradient. Various CA blends are nowadays commercially available to cover wide pH ranges for routine IEF analyses. Peak Broadening during Analyte Mobilization. It is important to emphasize that the extracted ion chromatogram (EIC)-MS profiles of proteins in OMJ-CIEF-MS experiments only partially reflect their actual in situ distribution in the fractionator. For example, the observed width of the EIC-MS profile for unfocused myoglobin (ca. 5 min, Figure 2) corresponds to ∼15 μL volume of protein band at the detection point, which is 7−8 times larger than the volume of the sample injection loop (2 μL). The protein band is significantly broadened during hydrodynamic mobilization, e.g., due to the interaction with capillary walls or sample diffusion.10,40 As can be seen from Figure 3, EIC peaks of analyzed proteins are broadening in the following order: myoglobin < cytochrome c < lysozyme. This trend reflects the increasing residence time of corresponding proteins in the separation capillary during the mobilization process. While myoglobin is focused at the downstream end of the separation device and is rapidly delivered to the MS, lysozyme has to pass the entire volume of the device (ca. 15 μL) before ESI-MS detection. Over this time, the focused protein band is substantially affected by the interaction with both mobile and stationary phases of the fractionator. In a reference experiment, 6859

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method. In this respect, selective mobilization can be viewed as in situ fraction collection, as if protein bands were “cut out” directly from the focal spot, like in gels or compartmentalized electrolyzers. In contrast to these techniques, however, OMJCIEF enables online and fully automated transfer of isolated fractions for hyphenated capillary-based MS or RPLC analyses. It is also important to note that the analyte transfer can be flexibly timed, increasing the capacity of OMJ-CIEF to integrate into multidimensional separation. pI Fractionation. IEF separation is commonly integrated into hybrid analyses as a first-dimension technique to reduce the complexity of biological samples. In proteomics studies, prefractionation of protein and peptide mixtures by pI greatly alleviates the identification process, especially with regard to the discovery of post-translational modifications (PTMs).58−64 PTMs can be difficult to reveal in both MS and LC dimensions if they have very little or even no effect on the m/z value and retention time, respectively. In contrast, PTMs can be both isolated and identified in the IEF domain based on measurable modification-specific shifts induced in pI values.61 The employment of IEF as a sample prefractionation technique has substantially increased over recent years owing to the progress in commercial IPG-IEF instrumentation, which has enabled fraction recovery in the liquid phase.65−68 Liquid-phase analyte recovery nicely fits into LC−MS workflows and obviates tedious, error-prone analyte extraction and cleanup procedures that are frequent in gel-based methods. The multiple-junction interface endues OMJ-CIEF with high potential for online analyte fractionation. The pH profile across the separation capillary can be conveniently tailored via pinpoint solvent exchange to emphasize specific pI regions, and the focused fractions can then be selectively mobilized. It is also worth mentioning that the possibility of external electrolyte introduction via membrane interfaces in OMJ-CIEF increases practical convenience of its operation. Analyte mixture can be directly loaded for analysis without chemical pretreatment, and pure water can be run as carrier solvent, eliminating possible carryover effects associated with changing the separation buffer. Figure 4 summarizes a series of experiments in which OMJCIEF was successfully employed to fractionate model protein mixture according to designated pI cutoff values. The analyte mixture contained seven proteins dissolved in water covering a broad pI range: β-lactoglobulin A (pI 5.3), hemoglobin A (pI 6.8), myoglobin (pI 6.9−7.3), α-chymotrypsinogen A (pI 9), ribonuclease A (pI 9.5), cytochrome c (pI 10−10.5), and lysozyme (pI 11.3). In the first experiment, 200 mM aqueous solution of histidine (His) was used as electrolytic buffer in all external reservoirs. This high buffer concentration was chosen to allow for the rapid diffusion of His through the Nafion membranes. Small amphoteric His molecules enter the separation volume, which is initially filled with pure water, and modify its pH to their pI value of 7.6. Unlike ionic buffers such as ammonium formate, His assumes zwitterionic form in solution, and its density profile is therefore much less sensitive to the external electrical field. Furthermore, the high buffering capacity of His around its pI affords the stability of established pH = 7.6, preventing the formation of gradients across the capillary. As a result, the entire capillary volume serves as a pIdivider wall with a narrow cutoff value. In the external field, proteins start to migrate along the fractionator, the direction being dependent on their net charge at pH 7.6. Proteins with pI < 7.6 will be negatively charged and will therefore migrate downstream toward the positive pole (Figure 1), while proteins

Figure 4. OMJ-CIEF separation of a model protein mixture in ampholytic His (left) and Arg (right) buffers. The analyte mixture contained seven proteins covering a broad pI range: β-lactoglobulin A (22 μM, pI 5.3), hemoglobin A (3 μM, pI 6.8), myoglobin (10 μM, pI 6.9−7.3), α-chymotrypsinogen A (31 μM, pI 9), ribonuclease A (58 μM, pI 9.5), cytochrome c (65 μM, pI 10−10.5), and lysozyme (56 μM, pI 11.3) dissolved in water. All the external reservoirs were filled with the same electrolyte buffer (200 mM aqueous His or Arg), and pure water was used as a carrier solution. IEF was carried over 1 h in the constant current regime (5 μA). All the fractions were mobilized for ESI-MS detection simultaneously via hydrodynamic pumping (3 μL min−1). For better visual perception EIC-MS profiles of the proteins are plotted separately and normalized to their peak intensity.

with pI > 7.6 will acquire positive charge and move in the opposite direction. As a result, the protein mixture will separate into acidic (pI < 7.6) and basic (pI > 7.6) fractions located at the opposite termini of the fractionator. Note that, due to the absence of electroosmotic flow in OMJ-CIEF, analytes are separated merely based on their charge (in other words, pI value), rather than size-to-charge ratio, as in CZE. Fractionation of the protein mixture in His buffer following the described approach is shown in Figure 4, left. The acidic fraction contained β-lactoglobulin A, hemoglobin A, and myoglobin, which are negatively charged in His buffer and therefore migrated downstream to the positive pole of the capillary. The basic fraction contained α-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme and was focused at the negative pole of the fractionator. Despite the notable peak broadening during analyte mobilization, the fractions arrived to the detection point without overlapping (Figure 4, left), which points to a large spatial cushion right after the focusing stage. Indeed, the acidic (or the basic) fraction could be selectively released when necessary via electrocapturing of the oppositecharged fraction. The use of single-component fractionation buffer, such as His, offers a number of benefits when CIEF is integrated into multiple-stage analyses. Compared to heterogeneous ampholytic blends, the presence of singular His is easier to discriminate against in both LC and MS domains, allowing for more reliable identification of peptides and proteins. Furthermore, His is a suitable buffer for tryptic digestion,69 and the released fractions can therefore be directly transferred 6860

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agreement with their predicted pI values (Figure 5). The negatively charged acidic fraction (pI < 5) drifted to the downstream terminus of the fractionator (RT ∼ 1.5 min), while the basic fraction (pI > 7.6) drifted upstream (RT ∼ 5 min). Interestingly, an intermediate fraction was formed (RT ∼ 3 min) containing peptides with pI values ∼6−7. Even though these peptides are predicted to be negatively charged in His buffer, their downward migration is probably hindered by more abundant acidic peptides (pI < 5) occupying the very end of the separation capillary. Note that both the peptide mixture and carrier solution already contained His (5 mM) necessary for IEF, unlike the experiments described in Figure 4, in which pure water was used. For this reason, it was not necessary to apply high His concentration in the external vials (5 mM in Figure 5 vs 200 mM in Figure 4).

online to an immobilized enzyme microreactor (IMER) without any purification steps. Whenever required, His can be efficiently removed from analyte solution using online dialysis interfaces with low molecular weight cutoff or chromatographic separation. Figure 4 (right column) illustrates IEF separation of the same protein mixture performed in arginine (Arg) buffer. Arg is an ampholytic electrolyte and, analogous to His, acts as a discriminator wall with a sharp cutoff value equal to its pI 10.8. This higher cutoff results in a different partition, allowing for the selective isolation of lysozyme, which is the most basic protein in the mixture. The selective removal of most basic proteins prior to ESI-MS analysis can sizably advance the ionization efficiency of other constituents in the analyzed mixture, which is often hindered in the presence of basic fraction. On the contrary, preisolation of basic proteins via removal of acidic fraction is of particular interest in top-down proteomics approaches relying on electron capture or electron transfer dissociation (ECD, ETD). Having the largest number of amino groups, these proteins get most abundantly charged in positive ion mode ESI-MS, which enables more comprehensive fragmentation by ECD and ETD compared to the acidic ones.70,71 For different cutoff values, an appropriate ampholyte can be chosen from a variety of natural and synthetic products, the major requirements being the low molecular weight to allow facile diffusion through Nafion junctions, high buffering capacity, and tolerance by the MS. For example, glutamic acid (pI 3.1) could be considered as an option to perform fractionation in the low-pH range. Peptide Separation. Unlike microdialysis interfaces with relatively high molecular weight cutoff values (∼3000 Da), Nafion membranes have considerably smaller pores, which allows analyses of smaller molecules, e.g. peptides, without analyte leakage. Supporting Information Figure S4 and Figure 5 demonstrate the results of OMJ-CIEF separation performed in 5 mM His buffer on a mixture of peptides produced by tryptic digest of bovine serum albumin (BSA). Altogether, 37 peptides covering more than 60% of the protein sequence were identified via searching the peptides’ MS/MS by Mascot. The observed OMJ-CIEF separation of analyzed peptides is in good

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CONCLUSION The growing popularity of recently introduced IPG-IEF fractionators (e.g., OFFGEL fractionator from Agilent) reflects the steady demand for carrying separation of biological samples in solution phase. Direct analyte recovery from solution precludes the pitfalls associated with gel methods, such as tedious and error-prone protocols for sample extraction and purification. Owing to the capillary format of operation, OMJCIEF combines the merits of solution-phase separation with the possibility of automated online sampling to MS, LC, CE, and other capillary-based analyses. Advantageous to traditional CIEF, the segmented interface of OMJ-CIEF enables in situ manipulation over the fractionation process. The chemical profile of the mobile phase can be tailored externally via pinpoint solvent exchange through Nafion junctions, and analytes focused in different segments of OMJ-CIEF can be mobilized separately in time. Performed in partitioned volume with autonomously controlled segments, OMJ-CIEF can be viewed as a capillary-based analogue of compartmentalized IPG-IEF electrolyzers. The good online compatibility of OMJCIEF with capillary-based separations renders it particularly promising in high-throughput proteomics as a first-dimension prefractionation unit orthogonal to LC and MS domains. Due to its high loading capacity (>100 pmol), the device can be useful in semi-online and off-line analyses as a preparative tool for analyte purification, fractionation, and preconcentration purposes. The high cost-efficiency and the ease of design and operation may eventually promote the adoption of OMJ-CIEF in a wide range of bioanalytical studies.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46 8 524 87594. Notes

Figure 5. OMJ-CIEF separation of tryptic BSA digest. For the CIEF analysis peptide mixture was prepared in 5 mM aqueous histidine. The same buffer system was used as a carrier solution and electrolytic liquid in external reservoirs. IEF was carried over 1 h in the constant current regime (5 μA). All the fractions were mobilized for ESI-MS detection simultaneously via hydrodynamic pumping at 3 μL min−1. Peak elution time for the analyzed peptides was obtained from their EIC-MS profile (Supporting Information Figure S4).

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from VINNOVA (Grant EuroStar 2010) and the Swedish Research Council (VR Grant 2011-3699) is gratefully acknowledged. 6861

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dx.doi.org/10.1021/ac3013016 | Anal. Chem. 2012, 84, 6856−6862