Anal. Chem. 2005, 77, 7131-7136
Microfluidic Electrocapture for Separation of Peptides Juan Astorga-Wells, Susanne Vollmer, Sam Tryggvason, Tomas Bergman, and Hans Jo1 rnvall*
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Separation is fundamental in analytical chemistry, since in many cases analytes need to be fractionated in order to be properly identified or quantified. In the case of polypeptides, liquid chromatography (LC), gel electrophoresis, and capillary electrophoresis (CE) have taken a central role because of high resolving power and the development of methods for combination of these techniques with mass spectrometry (MS).1-4 In this context, methodologies based on liquid separations have gained especial attention since they are more compatible with MS than gel-based separations because of the avoidance of gel-cutting and in-gel digestion procedures. In addition, liquid-based separations are being increasingly used in proteome analysis after the emergence of shotgun digestion of proteins, where several thousands of peptides need to be fractionated before peptide sequence analysis by tandem MS.5-7 Despite many advances, LC-MS instrumentation still remains expensive because of the necessary use of highpressure and nanoflow rate pumps, automated solvent gradients for elution, and on-line preconcentration prior to injection into the capillary column. CE is methodologically simpler, faster, and less costly than LC but has poor detection limits compared with the
latter technique. Different on-line preconcentrators have been successfully developed for the analysis of peptides by CE-MS (e.g., solid-phase extraction,8 membranes,9 and immunoadsorbents10). Using these improvements, CE-MS remains five times less sensitive than nanoLC-MS for peptide sequence analysis by tandem MS.11 Although these advances show the potential of CEMS, the lack of commercially available preconcentrators hinders its use. Thus, although LC and CE are complementary and cover the analysis of a wide range of molecules, there is still a need for novel separation techniques in the analysis of complex biological samples. In this report, we outline a separation strategy especially suitable for microsystems in the analysis of complex mixtures of peptides. The method is based on a device previously employed to immobilize charged molecules in a flow stream without using solid-phase materials or chemical binding. Immobilization is achieved through the counteracting effects of hydrodynamic and electric forces upon target molecules together with a membranestacking effect generated by differential ion transport between the fluidic channel and the electrode chambers.12,13 This methodology, “electrocapture”, has previously been shown to be capable of concentration, solvent exchange, sample cleanup (desalting and detergent removal), and performance of multistep microreactions (on-line reduction, alkylation, and trypsin digestion for MS analysis).12-16 We now extend this concept further, to peptide separations, introducing a novel principle using a voltage gradient for elution. The work was initiated by the observation that when negatively charged molecules were captured, acidic peptides were trapped at lower electric field strengths than less acidic peptides. It was also found that peptides with a pI higher than the running buffer (pH 8.0) were not captured and were observed in the first fractions collected at the outlet of the microfluidic device. Thus, since different peptides are captured depending on the electric field strength, could captured peptides be sequentially released using
* Corresponding author. Tel: +46-8-524 877 02. Fax: +46-8-337 462. E-mail:
[email protected]. (1) Yates, J. R., 3rd. Trends Genet. 2000, 16, 5-8. (2) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (3) Naylor, S.; Kumar, R. Adv. Protein Chem. 2003, 65, 217-248. (4) Jolles, P., Jo ¨rnvall, H., Eds.; Proteomics in Functional Genomics: Protein Structure Analysis; Birkha¨user: Basel, 2000. (5) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946951. (6) Wolters, D. A.; Washburn, M. P.; Yates, J. R., 3rd. Anal. Chem. 2001, 73, 5683-5690. (7) Storms, H. F.; van der Heijden, R.; Tjaden, U. R.; van der Greef, J. Electrophoresis 2004, 25, 3461-3467.
(8) Figeys, D.; Ducret, A.; Aebersold, R. J. Chromatogr., A 1997, 763, 295306. (9) Tomlinson, A. J.; Naylor, S. J. Capillary Electrophor. 1995, 2, 225-233. (10) Guzman, N. A.; Stubbs, R. J. Electrophoresis 2001, 22, 3602-3628. (11) Pelzing, M.; Neususs, C. Electrophoresis 2005, 26, 2717-2728. (12) Park, S.-R.; Swerdlow, H. Anal. Chem. 2003, 75, 4467-4474. (13) Astorga-Wells, J.; Swerdlow, H. Anal. Chem. 2003, 75, 5207-5212. (14) Astorga-Wells, J.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2003, 75, 52135219. (15) Astorga-Wells, J.; Bergman, T.; Jo ¨rnvall, H. Anal. Chem. 2004, 76, 24252429. (16) Astorga-Wells, J.; Vollmer, S.; Bergman, T.; Jo ¨rnvall, H. Anal. Biochem. 2005, 345 (1), 10-17.
A separation method based on electroimmobilization and sequential release of captured molecules is reported. A microfluidic electrocapture device is utilized to immobilize peptides in a microflow stream. After capture, the electric field is decreased in a stepwise manner, causing sequential release of the captured peptides according to their electrophoretic mobility. Tryptic peptides were separated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The separation power was high enough to increase the ionization yield of several peptides not seen in the unprocessed sample. In addition to separation, simultaneous sample cleanup was demonstrated for peptides obtained by shotgun tryptic digestion of membrane protein extracts.
10.1021/ac050931z CCC: $30.25 Published on Web 10/12/2005
© 2005 American Chemical Society
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Figure 1. Electrocapture device setup for separations.
a descending voltage gradient? The use of the electrocapture technology for separation purposes involves two steps. In a first step, charged molecules are captured by the system. Second, the electric field is decreased to allow sequential release of captured molecules. The principle is demonstrated by the separation of typtic peptides for subsequent analysis by MALDI-TOF MS. EXPERIMENTAL SECTION Reagents and Chemicals. Phosphorylase b, bovine serum albumin, β-casein, myoglobin, β-lactoglobulin, lysozyme, hemoglobin, carbonic anhydrase, cytochrome c, alcohol dehydrogenase (from yeast), dithiothreitol (DTT), iodoacetamide (IAA) and R-cyano-4-hydroxycinnamic acid were obtained from Sigma. TrisHCl 1 M, pH 8, ultrapure sterile solution was from United States Biochemical, porcine sequencing grade modified trypsin from Promega, and trifluoroacetic acid (TFA) from Applied Biosystems. The water used was from a MilliQ water purification system. Protein Solutions and Trypsin Digestion. The protein stock solutions were prepared at concentrations of 1-5 mg/mL. Trypsin digestion was performed in solution. Carbamidomethylation of cysteine residues was carried out by mixing 25 µL of protein stock solution with 5 µL of 45 mM DTT and subsequent incubation at 50 °C for 15 min. After cooling to room temperature, 5 µL of 100 mM IAA was added, and the mixture was further incubated for 15 min at room temperature. Following alkylation, 64 µL of TrisHCl buffer (pH 8) was added to a final concentration of Tris-HCl of 40 mM. For digestion, 1 µL of trypsin (1 µg/µL in 50 mM acetic acid) was added. The digestion was performed at 37 °C overnight, and the resulting tryptic peptides were stored at -20 °C. The digest was diluted with 40 mM Tris-HCl buffer (pH 8) before use. Preparation of Membrane Proteins. Tissue from rat kidney was washed in 500 µL of 12.5 mM NH4HCO3 and incubated on ice for 30 min. The sample was then subjected to freeze/thaw cycles (five times) in liquid nitrogen, sonicated four times for 10 s, and centrifuged for 5 min at 2000g, after which the supernatant was discarded. The pellet was resuspended in 500 µL of 12.5 mM NH4HCO3 and centrifuged at 6000g for 10 min. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 80000g for 30 min (Beckman Ti 70.1). The supernatant was discarded and the pellet dissolved in 25 mM NH4HCO3. The resulting sample was ultracentrifuged at 100000g for 1 h. The supernatant was discarded, and the pellet was resuspended in 1.5 mL of 100 mM Na2CO3, pH 11.3, and centrifuged at 13 000 rpm, 4 °C. The pellet was solubilized in 50 mM NH4HCO3, pH 7.9, 1% CHAPS, and was ready for in-solution digestion. For digestion, 1 µL of trypsin (1 µg/µL in 50 mM acetic acid) was added to 100 µL of 1:1 (v/v) mixture of sample with 80 mM Tris-HCl, pH 8.0, 7132
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containing 60% acetonitrile. The digestion was performed at 37 °C overnight and the digest stored at -20 °C. Mass Spectrometry and Crude Sample Analysis. All mass spectra were obtained with a Voyager DE-PRO MALDI time-offlight (TOF) mass spectrometer (Applied Biosystems) operated in the positive-ion mode. Reflector mode was used at 20-kV accelerating voltage, 74.5% grid voltage, 0.005% guide wire, and 200-ns delayed extraction. The instrument was calibrated with Sequazyme Peptide Mass Standard (Applied Biosystems). The matrix was R-cyano-4-hydroxycinnamic acid (saturated solution in 60% acetonitrile/0.1% TFA). The results from experiments using the microfluidic device were compared with those from unprocessed samples. Aliquots of 0.4 µL of crude sample were mixed 1:1 (v/v) with matrix solution, spotted onto the MALDI target, and analyzed. Device. The microfluidic device was manufactured using a piece of PEEK tubing (127-µm i.d. and 512-µm o.d.; Upchurch, Oak Harbor, WA) with two small openings made with a scalpel. The distance between the openings was 2.6 cm. They were covered with a conductive tubular cation-selective poly(tetrafluoroethylenesulfonate) membrane (Permapure Inc, Toms River, NY) with the dimensions 330-µm i.d. and 610-µm o.d.. The junctions were placed into separate electrode chambers made from 500-µL plastic tubes (Eppendorf, Hamburg, Germany) and filled with 40 mM Tris-HCl buffer (pH 8). Electrodes of platinum wire were placed into the electrode chambers. The anode was connected to a high-voltage power supply (Bertan, model ARB 30, Hicksville, NY), and the cathode was grounded. The current was monitored using a chart recorder, which was connected to the current monitor output of the power supply. A syringe pump (Harvard Apparatus, Hollistion, MA) equipped with a 100-µL gastight syringe (Hamilton, Reno, NV) that was filled with 40 mM TrisHCl (pH 8) provided a continuous hydrodynamic flow stream. The microfluidic device is shown in Figure 1. Injector Setup. The syringe pump and the microfluidic device were connected to a microinjector with a 1-µL internal loop. The loop was filled with sample using a homemade T-connection that utilizes an air-pressurized hydrodynamic displacement. Using this system, the loop can be filled directly from a plastic tube ensuring minimum sample manipulation. System Operation. The general operation involves injection, capture, voltage reduction step, and MALDI plate spotting. The device is filled with 40 mM Tris-HCl buffer using the syringe pump at a flow rate of 0.2 µL/min. The power supply is switched on, and the sample is injected into the system (in waste position). After ∼2 min, the system is switched to the inject position, and
Figure 2. Scheme of the electrocapture-based separation and sample preparation.
the sample is transported into the device via the flow stream at 0.2 µL/min created by the syringe pump. To ensure that the sample is entirely injected into the system, the initial voltage was kept for 45 min before starting the voltage reduction step. This period can be prolonged when sample cleanup is carried out, since the original solvent is exchanged for the background electrolyte. The initial voltage was decreased in steps of 5-10 V every min. Fractions corresponding to 0.2 µL (1 min) were spotted onto the MALDI plate and mixed with matrix solution (1:1). Samples and buffer solutions were degassed for 20 min before injection. A scheme of the electrocapture base separation is shown in Figure 2. Safety. Since the device works with voltages in the order of ∼500 V, proper safety precautions should be taken. It is highly recommended to use a power supply with maximum output current in the microampere range (e.g., power supplies for CE). Other power supplies can be used if an unattended and wellgrounded instrument is built. Serious injuries or death might occur if these instructions are not followed. RESULTS AND DISCUSSION Establishment of Separation Principle. To test whether the electrocapture device can be employed to fractionate polypeptides, samples containing tryptic peptides obtained from digestions of nine proteins were analyzed. The samples were injected (1 µL) while applying an electric field of 139 V/cm and a flow rate of 0.2 µL/min. To ensure that the whole sample plug has passed through the system, a 45-min delay was included after the sample injection. After this period, peptides are either captured or swept out from the system. Since the device captures peptides that have electrophoretic velocities higher than or equal to the velocity of the flow, peptides that have low electrophoretic mobility (or are uncharged) at the pH used will not be retained in the electrocapture device and, therefore, will appear in the first fractions as shown in Figure 3A. After the injection delay, the voltage was decreased in a stepwise fashion. As seen in Figure 3B-E, different profiles of peptides appear in each fraction obtained after voltage reduction, showing that the electrocapture device can indeed sequentially release captured polypeptides according to how strong they are captured. In theory, peptides with low electrophoretic mobility
(µe) need a higher electric field in order to be captured than those with high µe. A closer study of the separation principle is shown in Table 1, where the pI and electrophoretic properties of identified tryptic peptides were calculated using bioinformatics tools. A value directly correlated to µe was calculated, q/M2/3, where q is the charge and M is molecular weight of the polypeptide.17 Positively charged peptides were observed in the first fractions with pI values from 9.8 to 11.8. At lower electric field strengths, negatively charged polypeptides were eluted with pI and q/M2/3 values directly correlated with the applied voltage. As seen in Figure 3, peptides observed in the unprocessed sample can be seen in at least one of the electrocapturegrams (data graph of capture voltage, signal intensity versus m/z). Only one low-intensity peak was missed. In contrast, several peptide peaks can only be seen, in particular electrocapturegrams indicating that the separation was sufficient to increase the ionization yield of these species (14 peptide peaks). Using other separation methods, several studies have demonstrated that the analysis of samples containing complex mixtures of analytes is normally facilitated by a preceding separation step.18,19 Since the ionization by MALDI is a competitive process, molecules present at high concentration or those easily ionized by MALDI can suppress the ionization of other molecules present at low concentration or less prone to be ionized.20,21 Another example of separation can be seen in Figure 4 where 200 fmol of phosphorylase b peptides was analyzed. Because of the characteristics of the power supply used, the smallest voltage step possible was 9-15 V. Smaller voltage steps should considerably improve the separation efficiency, and a continuous gradient would presumably be optimal for resolution, as in chromatography separations. However, already at this stage (17) Rickard, E. C.; Strohl, M. M.; Nielsen, R. G. Anal. Biochem. 1991, 197, 197-207. (18) Covey, T. R.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63, 11931200. (19) Hoja, H.; Marquet, P.; Verneuil, B.; Lotfi, H.; Penicaut, B.; Lachatre, G. J. Anal. Toxicol. 1997, 21, 116-126. (20) Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich, F. Electrophoresis 1998, 19, 1910-1919. (21) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 41604165.
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Figure 3. MALDI-MS spectra of a tryptic peptide mixture in 40 mM Tris-HCl, pH 8, generated from nine proteins: β-casein (1.3 pmol), myoglobin (4.6 pmol), β-lactoglobulin (1.7 pmol), lysozyme (6.5 pmol), hemoglobin (1.2 pmol), carbonic anhydrase (2.8 pmol), cytochrome c (1.5 pmol), alcohol dehydrogenase (yeast, 2.8 pmol), and phosphorylase b (0.5 pmol). The sample was injected at a flow rate of 0.2 µL/min and at an initial electric field of 139 V/cm. The peptides were captured for 45 min after which the voltage was decreased in steps of 9-15 V. Eluates were analyzed by MALDI-MS. Arrows in the electrocapturegrams show peptide peaks that were not detected in the corresponding unprocessed sample.
the present modality with stepwise changes demonstrates the feasibility of electrocapture-based separations. Online Sample Preparation and Separation. In previous studies, we have shown the utilization of the electrocapture technology as a sample preparator device.14,15 In addition to multistep microreactions, solvent exchange, preconcentration, and desalting, the electrocapture device can be used to remove detergents from samples containing peptides and proteins. In this context, we now explored the possibility to carry out on-line combined separation and cleanup of peptide samples obtained by shotgun digestion of membrane protein fractions from rat kidney cells. Sample cleanup was carried out by extending the capture 7134 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
period to 1 h after injection, by which sample solvent is changed for the running electrolyte. It must be pointed out that the removal of CHAPS detergent is critical for obtaining mass spectrometric signals from peptides.14,15 Successful sample cleanup and separation was obtained from digests of membrane protein preparations, revealing many peaks in the MALDI mass spectra. Since each signal is represented by three vectors (intensity, m/z, electrocapture voltage), a single sample run can be displayed in a three-dimensional electrocapturegram, in which the overall separation can be seen (Figure 5). The presence of acetonitrile in the washing solution was found to be critical for detergent removal. This effect is likely caused
Table 1. Molecular Properties of Identified Tryptic Peptides Separated and Recovered from the Protein Mixture Digesta electric field (V/cm) 130 92
67
62
39 a
sequence
m/z
pI
q/(M2/3)
TGPNLHGLFGR (cytochrome c) AVPYPQR (casein) SHHWGGYGK (carbonic anhydrase) KTGQAPGFTYTDANK (cytochrome c) SISIVGSYVGNR (ADH) HNGPEHWHK (carbonic anhydrase) DYYFALAHTVR (phosphorylase b) HGLDNYR (lysozyme) IVSDGNGMNAWVAWR (lysozyme) FESNFNTQATNR (lysozyme) DGPLTGTYR (carbonic anhydrase) GTDVQAWIR (lysozyme) LHVDPENFR (hemoglobin) SIGGEVFIDFTK (ADH) EALDFFAR (ADH) VEADIAGHGQEVLIR (myoglobin) VNVDEVGGEALER (hemoglobin)
1168.62 830.45 1013.00 1598.76 1251.67 1141.53 1355.63 874.40 1675.80 1428.65 979.48 1045.54 1126.50 1312.68 968.50 1606.85 1386.44
11.8 9.8 9.8 9.8 9.8 8.0 7.8 7.8 6.7 6.9 6.8 6.7 5.1 4.0 4.0 4.3 3.8
+9.0 × 10-3 +11 × 10-3 +9.9 × 10-3 +9.1 × 10-3 +8.6 × 10-3 0 -4.1 × 10-4 -5.5 × 10-4 -7.1 × 10-4 -7.9 × 10-4 -10 × 10-4 -19 × 10-4 -92 × 10-4 -92 × 10-4 -120 × 10-4 -150 × 10-4 -233 × 10-4
Values of pI and q/M2/3 drop off fairly well correlated with the electric field.
Figure 4. MALDI-MS spectra of 200 fmol of tryptic peptides of phosphorylase b in 40 mM Tris-HCl, pH 8. The sample was injected into the system at a flow rate of 0.2 µL/min. The peptides were captured for 30 min at an initial electric field of 98 V/cm and then sequentially released by reducing the voltage in steps of 9-15 V.
by the necessity to exchange detergent molecules attached to hydrophobic peptides for other molecules of low polarity, such as acetonitrile. CONCLUSION The reported methodology shows a novel separation strategy, possible to combine with on-line sample preparation. It has the potential to become a proteomics tool for prefractionation or separation in the analysis of complex peptide mixtures. This
assessment is based on three main points. First, the separation power is high enough to considerably increase the ionization of many peptides. Second, it can handle samples at the femtomole level. And third, the possibility to carry out on-line separation and cleanup of complex biological samples, such as membrane protein digests, is demonstrated. The electrocapture-based separations might have several advantages in comparison to standard technologies. Compared to ion-exchange chromatography, the electrocapture-based separaAnalytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 5. Three-dimensional electrocapturegram of tryptic peptides obtained from shotgun digestion of membrane proteins extracts (rat kidney). The sample was injected into the system in 40 mM Tris-HCl, pH 8, with 30% acetonitrile at a flow rate of 0.2 µL/min and at an initial voltage of 138 V/cm. The sample was captured for 1 h. The voltage was then decreased in steps of 9-15 V, and eluates were analyzed by MALDI-MS. Numbers on the z-axis correspond to fractions collected at different electric field strengths, where 0-12 correspond to 138, 132, 126, 115, 109, 103, 98, 92, 86, 80, 75, 69, and 63 V/cm, respectively.
tions do not use salt gradients, making it possible to directly analyze the fractions without the otherwise necessary desalting procedure before MS analysis. Furthermore, the technique simplifies the design of the analytical system since the gradient is made by the power supply and not by a high-pressure pump. In respect to CE, electrocapture-based separations can handle microliterrange samples through commercially available injectors, in contrast to the nanoliter-range injections used in CE analysis. Also, since the system uses continuous pumping, fraction deposition onto the MALDI target becomes an uncomplicated issue and suitable for direct connection to microspotters. To assess the real potential and limits of this novel separation tool, further studies
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need to evaluate the resolution and recovery of the analytical process. ACKNOWLEDGMENT This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, Vinnova, the European Commission (LSHC-CT-2003-503297), the Wallenberg Consortium North, and Karolinska Institutet. Received for review May 27, 2005. Accepted September 2, 2005. AC050931Z
