Multistep Microreactions with Proteins Using Electrocapture Technology

disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and tryps...
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Anal. Chem. 2004, 76, 2425-2429

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Multistep Microreactions with Proteins Using Electrocapture Technology Juan Astorga-Wells, Tomas Bergman, and Hans Jo 1 rnvall*

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

A method to perform multistep reactions by means of electroimmobilization of a target molecule in a microflow stream is presented. A target protein is captured by the opposing effects between the hydrodynamic and electric forces, after which another medium is injected into the system. The second medium carries enzymes or other reagents, which are brought into contact with the target protein and react. The immobilization is reversed by disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and trypsin digestion of proteins is demonstrated and was monitored by MALDI mass spectrometry. Microfluidic devices promise to revolutionize chemical analysis, giving fast, portable, and inexpensive assays that could have an impact on any field in which a chemical analysis is needed.1-3 Despite recent progress, standard procedures routinely performed in “macro” devices or laboratory tubes are difficult to carry out in micrometer size structures. For example, microscale methodologies based on adsorption or immobilization, such as packing beads,4,5 membranes6,7 or hydrogels,8-10 are difficult to manufac* Corresponding author. Phone: +46-8-524 8 7702. Fax: +46-8-337 462. E-mail: [email protected]. (1) Beebe, D. J.; Mensing, G. A.; Walker, G. M. Annu. Rev. Biomed. Eng. 2002, 4, 261-286. (2) Chovan, T.; Guttman, A. Trends Biotechnol. 2002, 20, 116-122. (3) Huikko, K.; Kostiainen, R.; Kotiaho, T. Eur. J. Pharm. Sci. 2003, 20, 149-171. (4) Xie, B.; Mecklenburg, M.; Danielsson, B.; O ¨ hman, O.; Norlin, P.; Winquist, F. Analyst 1995, 120, 155-160. (5) Davis, M. T.; Lee, T. D.; Ronk, M.; Hefta, S. A. Anal. Biochem. 1995, 224, 235-244. (6) Tomlinson, A. J.; Braddock, W. D.; Benson, L. M.; Oda, R. P.; Naylor, S. J. Chromatogr., B 1995, 669, 67-73. (7) Gao, J.; Xu, J.; Locascio, L. E.; Lee, C. S. Anal. Chem. 2001, 73, 26482655. (8) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (9) Seong, G. H.; Zhan, W.; Crooks, R. M. Anal. Chem. 2002, 74, 3372-3377. (10) Zhan, W.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2002, 74, 4647-4652. 10.1021/ac0354342 CCC: $27.50 Published on Web 04/01/2004

© 2004 American Chemical Society

ture in micrometer-sized channels and are prone to failure from clogging and solvent incompatibilities. In this context, we have focused on a new microfluidic approach that immobilizes charged molecules in a flow stream without chemical bonding or solid supports. This “capture device” or “electrocapture device” utilizes an electric field to capture charged molecules traveling in a flow stream.11-13 The electric field counteracts the hydrodynamic sweeping force to capture charged molecules attracted toward the upstream electrode. As long as the electric field is connected, the device traps charged molecules passing through the nanoliter-volume capture zone. After capture and buffer replacement, the electric field is disconnected, and the molecules are released for on-line or off-line analysis. The first use of the capture device was for on-line preconcentration and desalting of crude DNA products from sequencing reactions before capillary electrophoresis.11 The negatively charged DNA fragments were captured and concentrated between the electrified junction zones, and desalting was performed by hydrodynamic washing. In a first report on applications to proteins, the capture device was used as an off-line preconcentrator device for capillary electrophoresis.12 In this case, a mixture of negatively charged proteins was captured and concentrated at the same spot. More than 10 µL of sample was concentrated into a nanoliter volume capture zone and loaded into the separation capillary, giving at least a 40-fold signal enhancement compared to a normal injection. In another report, the electrocapture device was used to carry out sample preparation of tryptic peptides and proteins in salt- and detergent-containing solutions before peptide mass mapping and protein analysis by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS).13 In this case, polypeptides were captured in their positively charged state. Sample cleanup was performed by buffer exchange. (11) Park, S.-R.; Swerdlow, H. Anal. Chem. 2003, 75, 4467-4474. (12) Astorga-Wells, J.; Swerdlow, H. Anal. Chem. 2003, 75, 5207-5212. (13) Astorga-Wells, J.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2003, 75, 52135219.

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Figure 1. Schematic representation of the electrocapture device to perform microreactions. For details of construction and operation, see text. A PEEK tubing with two gaps covered by conductive membranes was connected via a six-port valve to two syringes. The syringe pumps provide the injection of different solutions.

In the present report, we describe yet another principle for the use of the electrocapture device, to carry through microreactions. The capture zone is utilized as a microreactor through electroimmobilization of at least one of the reactants in the nanoliter volume chamber. For this work, we have chosen key enzymatic reactions in proteomics, i.e., trypsin digestion, reduction, alkylation, and subsequent direct identification by peptide mass mapping by MALDI-MS. Since most proteins are negatively charged at the pH where the proteolytic reaction is optimal (pH 8-9), we first investigated if a target protein and trypsin could be co-captured and react at the capture zone. After successful microdigestion experiments, we also found that reduction, alkylation and detergent removal could be carried out by using the same principle. Combined, this leads to a protocol in which the capture device is used for microreactions involving protein pretreatment (reduction and alkylation), sample cleanup, cleavage, and product identification by mass spectrometry. EXPERIMENTAL SECTION Reagents and Chemicals. Horse muscle myoglobin (Mb), bovine serum albumin (BSA), β-casein, dithiothreitol (DTT), iodoacetamide, and R-cyano-4-hydroxycinnamic acid were obtained from Sigma Chemical Co. (St. Louis, MO). β-Lactoglobulin was from Applied Biosystems (Foster City, CA). Porcine, modified trypsin (sequencing grade) was from Promega (Madison, WI), and trifluoroacetic acid (TFA) was from Applied Biosystems (Warrington, U.K.). The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) detergent was obtained from Pharmacia Biotech (Uppsala, Sweden). The water used was from a Milli-Q water purification system (Millipore). Solutions. Acetonitrile-containing solutions were used in order to accelerate the digestion rate of trypsin.14 Unless otherwise specified, all proteins (including trypsin) were dissolved in the washing buffer, which consisted of 50 mM Tris-HCl (pH 8.0) in 50% acetonitrile (v/v). For on-line digestion, a solution of 10 ng/ µL trypsin was used. Mass Spectrometry. All mass spectra were obtained with a Voyager DE-PRO MALDI-time-of-flight (TOF) mass spectrometer (Applied Biosystems) operated in the positive ion mode. Reflector mode was used at 20-kV accelerating voltage, 71% grid voltage, 0.005% guide wire voltage, and 180-ns delayed extraction. The matrix was R-cyano-4-hydroxycinnamic acid (almost saturated solution in 70% acetonitrile/0.1% TFA). Database searches employed the MS-Fit software (http://prospector.ucsf.edu) and the SwissProt protein sequence database. (14) Russell, W. K.; Park, Z. Y.; Russell, D. H. Anal. Chem. 2001, 73, 26822685.

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Device Fabrication. The microfluidic device was manufactured as schematically outlined in Figure 1. Two small openings were made on a piece of PEEK tubing (Upchurch, Oak Harbor, WA) having the dimensions 127-µm i.d. and 512-µm o.d., and the distance between the gaps was 1.6-cm. The openings were covered with a conductive tubular cation-selective membrane of poly(tetrafluoroethylensulfonate) material (Permapure Inc, Toms River, NY) having the dimensions 330-µm i.d. and 610-µm o.d. The two electrical junctions were placed in separate electrode chambers made from 500-µL plastic tubes (Eppendorf, Hamburg, Germany) filled with 50 mM Tris-HCl (pH 8.0). Electrodes of platinum wire were placed into the electrode chambers. The anode was connected to a high-voltage power supply (Spellman, Plainview, NY), and the cathode was grounded. Current was monitored using a homemade current-to-voltage converter circuit digitally converted by a PCMCIA acquisition card (CyberResearch, Branford, CT) employing a Notebook-PC with Labtech software (Laboratory Technologies Co., Wilmington, MA). To produce a continuous hydrodynamic flow stream and to perform sample and reagent injections, two syringe pumps (Harvard Apparatus, Holliston, MA) equipped with a 500-µL gastight syringe (Hamilton, Reno, NV) were connected to the capture device through a sixport valve (Upchurch, Oak Harbor, WA). The entire system was mounted on an electrically grounded breadboard. Safety Considerations. Since the device works with voltages in the range of ∼400 V, proper safety precautions should be taken when the high voltage is on. It is strongly recommended to use a power supply with maximum output current in the microampere range (e.g., power supplies for capillary electrophoresis). To avoid electrical shock, current values should be measured to ensure the electrical continuity of the fluidic channel. System Operation. Voltage and flow rate values were obtained from previous work.12 Unless otherwise stated, all experiments were performed at a flow rate of 0.3 µL/min and an electric field of 180 V/cm. The general operation of the microfluidic electrocapture device in this proteomic application involves five steps, of which steps 2-4 constitute the 3-step microreaction: (step 1) injection of the sample and capture of target protein, (2) reduction of disulfide bonds by injection of DTT, (3) alkylation of cysteine residues by injection of iodoacetamide, (4) injection of trypsin and proteolytic digestion, followed by (5) release of the products. In step 1, one of the two syringe pumps was loaded with washing solution (washing port) and the other, with the target protein solution (injection port). The device was filled with the washing solution, and the power supply was turned on. To start injection of the target protein, the valve was switched to the injection port. Once the sample was injected, the valve was

Figure 2. MALDI-TOF spectra of on-line microdigestion of BSA. A 15-pmol fraction of the sample containing alkylated BSA was injected and captured, followed by the injection of 1 pmol of trypsin. The incubation time after trypsin injection was 45 min. (A) Spectrum with the capture device voltage turned on. Sequence coverage of BSA was 31%, within 0.1 Da mass accuracy. (B) Spectrum from the same experiment but without application of voltage. Asterisks denote mass peaks corresponding to BSA tryptic peptides.

switched back to the washing port position. For microreactions in steps 2, 3, and 4, with the valve on the washing position, the syringe connected to the injection port was changed for a syringe containing 100 mM of DTT (for reaction 2), 45 mM of iodoacetamide (for reaction 3) and trypsin (for reaction 4) solutions. Reagents were injected by repeating the valve-switching procedure, during which each step finished with the valve in the washing position. Solutions were injected for ∼10 min at 0.3 µL/ min. For incubation in step 5, the valve was left in the washing position. Finally, once the reaction was completed, products were released by disconnecting the high-voltage power supply. Fractions corresponding to ∼0.4 µL were directly spotted onto the MALDI target plate and mixed with matrix (1:1, v/v). The protocol was modified for proteins that do not contain disulfide bonds where reduction (step 2) and alkylation (step 3) were not included.

RESULTS AND DISCUSSION Single Step Microreaction. We first tested the feasibility to carry through enzymatic reactions in the capture device. To investigate this, 15 pmol of alkylated BSA was injected and captured, followed by injection of 1 pmol of trypsin. Proteins were incubated in the device for 45 min. The electric field was disconnected, and the released band was directly spotted onto the MALDI-MS plate. Trypsin digestion was found to have occurred, and peptides with masses matching those from conventional digestion of BSA were observed (Figure 2A). To exclude the possibility that just unspecific binding of the target protein, trypsin, or both to the channel walls was responsible for the appearance of tryptic peptides, the same experiments without application of the electric field were carried through. Peptide peaks were then absent in the mass spectra, demonstrating that co-capture of BSA and trypsin is responsible for bringing the reactants together (Figure 2B). Further experiments were carried out to optimize the incubation time. We found that when using 10 pmol of both Mb and alkylated BSA, only 20 min was sufficient to obtain cleavage. However, the incubation time had to be increased to obtain sufficient amount of peptides when less than 10 pmol of Mb or alkylated BSA was injected. The lowest amount of protein loaded into the capture device that still gave positive peptide identification with Mb and 1 h incubation time was found to be on the order of 1 pmol (Figure 3). Lower protein levels should be possible to analyze by increasing the incubation time and, hence, the fragment yield. Furthermore, dead volumes between the syringes and the device can be decreased, which would lower protein adsorption and, hence, also increase the fragment yields. The volume of the microchamber was estimated using data from a study in which colored proteins were photographed while being captured.12 The protein band is a cylinder of ∼256-µm radius and 125-µm length, which corresponds to an ∼26-nL-volume chamber. At the voltages and flow rate values used in these experiments, the target protein and trypsin are, therefore, likely to be located within this small volume. Consequently, the reactions are carried out at a protein concentration down to 1 mg/mL (1

Figure 3. MALDI-TOF spectrum from on-line microdigestion of Mb. A solution of 1 µM Mb was injected for 4.5 min at 0.3 µL/min (1.3 pmol injected) and captured, followed by 10-min injection of the washing buffer. After that, a 0.7 µM trypsin solution was injected for 9.5 min (1.5 pmol injected) and captured. Captured proteins were incubated for 1 h and then spotted onto the MALDI-MS target and mixed 1:1 (v/v) with matrix solution. Asterisks denote mass peaks corresponding to Mb tryptic peptides.

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Figure 4. MALDI-TOF spectra from on-line reduction, alkylation and trypsin digestion of β-lactoglobulin and BSA. (A) β-lactoglobulin (5 pmol/ µL) was injected for 10 min and captured (15 pmol injected). The DTT, iodoacetamide, and trypsin solutions were sequentially injected for 10 min, each followed by a 40-min incubation period. (B) The same protocol was followed for BSA digestion (14 pmol injected), but in this case, the incubation time for digestion was 30 min. Products were spotted onto the MALDI target and mixed 1:1 (v/v) with matrix solution. Asterisks indicate the mass peaks corresponding to peptides that match with the database search. The symbol “C” indicates peptides that match with the theoretical masses of carbamidomethylated peptides.

pmol/26 nL), hence, at a range similar to that of traditional macroscale work. Multistep Microreactions. The possibility to carry out multistep microreactions was investigated with the two-step alkylation reactionsdisulfide bond reduction with DTT and the subsequent carbamidomethylation with iodoacetamidesbefore trypsin digestion. It should be noted that at the capture zone, the reaction can occur in two manners: reactants can be co-captured in the same spot as the target protein and react (as with trypsin), or they can pass through the capture zone and react by molecular collisions with the electroimmobilized protein. In this particular reaction, we expect that DTT and iodoacetamide react with the captured protein during passage through the capture zone, since their molecular weights and structures predict an electrophoretic behavior different from that of the negatively charged proteins. After injection and capture of β-lactoglobulin, plugs of DTT, iodoacetamide, and trypsin solutions were sequentially injected. β-Lactoglobulin contains two disulfide bonds. Efficient alkylation of half-cystine residues of β-lactoglobulin was obtained (Figure 4A). The fact that it is impossible to carry through MALDI-MS analysis of samples containing DTT and iodoacetamide at the levels used in these experiments demonstrates that both reagents 2428

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were removed and, indeed, passed through the capture zone, rather than being co-captured. Another on-line alkylation experiment was performed with a more complex protein, BSA, which has 35 cystine residues in 17 disulfide bonds. On-line reduction and alkylation, as well as trypsin digestion, was performed with 14 pmol of BSA. As seen in Figure 4B, successful alkylation and digestion of BSA was obtained. Peptide masses corresponding to the carbamidomethylated peptides were clearly observed in the spectra. Sample Cleanup. In a previous study,13 we showed that the capture device can be used for sample cleanup by buffer exchange and hydrodynamic washing. In the present report, we tested if sample cleanup could be performed via hydrodynamic washing while the trypsin solution and incubation buffer are injected. It should be pointed out that in the previous report,13 the polypeptides were captured and cleaned in their positively charged state (pH ∼3), whereas now, the proteins are negatively charged (pH 8.0). A solution of β-casein containing 50 mM Tris-HCl (pH 8.0) and 10 mM CHAPS detergent was captured, followed by injection of trypsin and incubation solutions, both containing 30 mM TrisHCl in 50% acetonitrile. Samples treated by the capture device were cleaned from the detergent, resulting in high-quality MALDI-

in 0.1% SDS, sample cleanup was not required in this application but would be necessary for protein mass spectrometric analysis.

Figure 5. MALDI-TOF spectra after on-line sample cleanup and microdigestion of β-casein. A solution of β-casein (1 pmol/µL) containing 50 mM Tris-HCl and 10 mM CHAPS detergent was analyzed. (A) A 100-µL portion of the protein solution was mixed with 1 µL of 1 µg/µL of trypsin and incubated at 37 °C for 1 h. Then 0.5 µL of the crude sample was mixed 1:1 (v/v) with matrix solution and spotted onto the MALDI target for analysis. (B) A 5-pmol fraction of the sample containing β-casein was treated with the capture device, allowing simultaneous on-line sample cleanup and trypsin digestion.

MS spectra (Figure 5B), while digestion of the crude solution did not give any recognizable peptide peaks because of signal suppression caused by CHAPS (Figure 5A). For analysis of digests

CONCLUSIONS This methodology solves many critical problems related to microfluidic reactions. Mixing, incubation, and sample pretreatment can be performed on-line. The lack of a solid support or chemical bonding for immobilization of proteins in a microflow stream opens a fast and versatile route to carry out other microreactions and assays. In addition, since proteins are retained by electric and not physical interactions, cross-contamination between samples is minimized. In fact, the same device was used to perform all the experiments in this report and in many others without a sign of cross contamination or carry over. Furthermore, since the major technological innovation is the presence of two ion-selective areas in the microfluidic channel, its integration into microchip format for large-scale production can be more straightforward than present technologies based on solid supports. ACKNOWLEDGMENT This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, Vinnova, and Karolinska Institutet.

Received for review December 4, 2003. Accepted February 26, 2004. AC0354342

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