Microchip Reactor Packed with Metal-Ion Chelated Magnetic Silica

Microchip Reactor Packed with Metal-Ion Chelated Magnetic Silica Microspheres for Highly Efficient Proteolysis Yan Li,† Xiuqing Xu,† Bo Yan, Chunhui Deng,* Wenjia Yu, Pengyuan Yang, and Xiangmin Zhang* Department of Chemistry & Research Center of Proteome, Fudan University, Shanghai 200433, China Received October 23, 2006

Abstract: An easily replaceable and regenerable protease microreactor with metal-ion chelated adsorption of enzyme has been fabricated on chip. Magnetic microspheres with small size (∼200 nm in diameter) and strong magnetism were synthesized and were modified with tetraethyl orthosilicate. The metal chelating agent of iminodiacetic acid was then reacted with glycidoxypropyltrimethoxysilane before its immobilization onto the surface of magnetic silica microspheres (MS microspheres). The metal ion of copper and enzyme were subsequently adsorbed onto the surface. The prepared MS microspheres were then locally packed into the microchannel by the application of a strong magnetic field using a magnet to form an on-chip enzymatic microreactor. Capability of the proteolytic microreactor was demonstrated by cytochrome c and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF/TOF MS with sequence coverage of 77% and 21% observed, respectively. This microreactor was also applied to the analysis of one RPLC fraction of rat liver extract. After a database search, 23 unique peptides corresponding to 7 proteins were identified when one RPLC fraction of rat liver extract was digested by the microreactor. This opens a route for its future application in top-down proteomic analysis. Keywords: magnetic silica microsphere • immobilized trypsin • on-chip microreator • metal-ion chelation

Introduction The advent of genomics and proteomics has increased considerably the need for fast, low-cost, and automated tools from protein analysis. Proteolysis is the key step for positive protein sequencing in proteomics research integrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide mapping and sequencing of proteins require as complete as possible fragmentation of the protein in a short time, resulting in well-defined and reproducible peptide patterns.1,2 The conventional techniques of in-solution digestion of proteins offer limited sensitivity and are time-consuming * To whom correspondence should be addressed. E-mail: [email protected] (C.D.); [email protected] (X.Z.). Tel.: +86-216564-3983. Fax: +86-21-6564-1740. † These two authors contributed equally to this work. 10.1021/pr060558r CCC: $37.00

 2007 American Chemical Society

procedures, affecting severely the determination of comprehensive proteome profiles.3,4 On the contrary, the immobilized enzyme has been developed for characterization of proteins with benefits from the reusability and stability of enzyme,5,6 the higher digestion efficiency for analyzing proteins, and no autolysis products.7-10 Microfluidic devices are becoming powerful tools for performing chemical or biological assays over the last several years, due to their high analysis speed and throughput, reduced reagent consumption, and improved separation performance.1,11 Several investigators have also begun to explore the role that enzymes could incorporate within a microchannel to form a microbioreactor to carry out highly efficient and low-level protein digestion. Relative to free enzymes in bulk solution, the enzymes immobilized in a microchannel are more stable and resistant to environmental changes, and they provide molecular-level interactions with flowing substrates. The heterogeneity of the immobilized enzyme systems allows multiple reuses of the enzymes, continuous operation of enzymatic processed, and a greater variety of bioreactor designs. Implementation of enzymatic reactions in microchannels allows a decrease of the amount of consumables and sample by several orders of magnitude. Detection sensitivity can also be improved for a sample with small total volume since no dilution is necessary, and the smaller scale increases the speed of diffusion-limited reactions12-15 allowing faster assays16,17 Finally, microfluidic integration may allow the long-term higher automation and better reproducibility.18 Several principles for the implementation of microfluidic enzymatic assays, such as physical adsorption, sol-gel encapsulation, and covalent linking have been explored.19-24 The sorption or covalent coupling of enzyme molecules to the inner surface of the chip is limited by the low specific inner surface of the microchannel and the impossibility to renew the enzyme activity. This limitation was overcome by the immobilization of enzyme molecules to the surface of nano/microparticles, such as silica beads,25 agarose beads,26 and zeolite nanoparticles.27,28 Particles were fixed in the channel by weirs, frits, or membranes,29 but the reproducible filling of the column remains a challenge. The use of porous polymer monolith is an efficient method for solving these complications,30-34 in which the diffusion resistance during mass transfer has been proved quite small and proteins could be easily coupled to the monolithic supports by the modification of the epoxide groups on the carriers. However, it requires elaborate in-capillary chemistry and leads to nonrenewable matrixes. Recently, magnetic spherical particles with micro- and nanometer size Journal of Proteome Research 2007, 6, 2367-2375

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technical notes

Microreactor with Metal-Ion Chelated MS Microspheres

Figure 1. Process of Cu-IDA-GLYMO-MS microspheres preparation and trypsin immobilization

are gaining an increasing attention35-37 due to their ease of manipulation and recovery. The use of magnetic spherical particles, which does not require frits and allows reversible immobilization of the matrix, is shown to have important potential in microfluidic device.12,14 The enzyme can be irreversibly immobilized onto the magnetic spherical particles by covalent bonding,37,38 but the regeneration of the prepared reactor is limited when the activity of the bound enzyme is damaged or destroyed.39 Metal-ion chelated immobilization of the enzyme is quite different from conventional approaches, and the enzyme is bound to the support media based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid, which is chemically bound onto the matrix. Thus, the enzymes could be removed with EDTA elution for regeneration of the support media.8 Recently, Zou40 et al. successfully developed an on-column enzyme miroreactor based on metal copper-ion chelated immobilization of trypsin, and it was applied to the peptide mapping analysis of proteins by MALDI-TOF MS. In this study, we initially prepared an easily replaceable and regenerable protease microreactor for microfluidic device by synthesis of the metal-ion chelated magnetic silica microspheres (MS microspheres) following the immobilization of enzyme and then organized by an external magnetic field in the channel of the µ-chip. To confirm the possibility of applying the µ-chip immobilized magnetic enzyme reactor for effective protein digestion, it was first utilized for peptide mapping analysis of cytochrome c (Cyt-C) and bovine serum albumin (BSA) with MALDI-TOF MS and further applied to analyze the reversed-phase liquid chromatography (RPLC) fraction of rat liver extract. 2368

Journal of Proteome Research • Vol. 6, No. 6, 2007

Experimental Section Materials and Chemicals. Bovine serum albumin (BSA, fraction V) was obtained from Bio Basic Inc. (Toronto, Canada). 3-Glycidoxypropyltrimethoxysilane (3-GLYMO), iminodiacetic acid (IDA), (TPCK)-treated trypsin, cytochrome c (EC 232-7009) and fluorescein isothiocyanate conjugate bovine serum albumin (FITC-BSA) were purchased from Sigma Chemical (St. Louis, MO). Fused silica capillary with 250 µm ID/380 µm OD for RPLC was purchased from Yongnian Optical Fiber Factory (Yongnian, Hebei, China). Packing materials of C8 particles, 5 µm Zorbax 300SB, from Agilent Technologies (Waldbronn, Germany) and spherical silica gel, Zorbax, BP-SIL, from DuPont (Wilmington, DE) were used for capillary HPLC (cHPLC) column making. Trifluroacetic acid (TFA) was purchased from Merck (Darmstadt, Germany); acteonitrile was HPLC grade from Fisher Scientific (Fairlawn, NJ). Water was purified using a Milli Q system (Millipore, Molsheim, France). All of the other chemicals were of analytical grade and used as received. Glass chips used in this work were obtained from Zhejiang University with one single channel of 20 (l) × 2 (w) × 0.15 (h) in mm. Preparation of MS Microspheres. The magnetic microspheres were synthesized through solvothermal reaction described as follows: 2.70 g of FeCl3‚6H2O was first dissolved in 100 mL of ethylene glycol under magnetic stirring. A yellow clear solution was obtained after stirring for 0.5 h. Then 7.20 g of NaAc (sodium acetate) was added to this solution. After being stirred for another 0.5 h, the resultant solution was transferred into a Teflon-lined stainless-steel autoclave with capacity of 200 mL. The autoclave was sealed and heated at 200 °C for 8 h and cooled to room temperature. The black magnetic microspheres were collected with the help of a magnet, followed by

technical notes

Li et al.

Figure 2. (A) Schematic representation of on-chip protocol for protein digestion and identification coupled with MALDI-TOF MS/MS. (B) Amplified on-chip microreactor within the microchannel. (C) Amplified metal-ion chelated MS microspheres for trypsin immobilization

washing with recycle of ethanol and deioned water for six times. The product was then dried in vacuum at 60 °C for 12 h. In order to obtain core-shell MS microspheres with narrow size distribution and uniform thickness of silica via sol-gel approach, magnetic microspheres (0.01 g) were first treated in HCl aqueous solution (5.0 mL, 2 M) under ultrasonic vibration for 5 min. Then, the microspheres were thoroughly washed with deioned water and redispersed in a mixture of ethanol (70.0 g), deioned water (20.0 g), and concentrated ammonia aqueous solution (1.0 g, 28 wt %) with the help of ultrasonication, and a stable dispersion was obtained. Subsequently, tetraethyl orthosilicate (0.05 g) was added to the above dispersion under mechanistic stirring, and the reaction was allowed to proceed for 12 h. Finally, by the use of a magnet, the product was separated, washed with ethanol and water, and then vacuumdried at 60 °C for 24 h. Modification of MS Microspheres for Enzyme Immobilization. In a 100 mL flask bottle, iminodiacetic acid (IDA, 4.20 g) was dissolved in sodium hydroxide aqueous solution (10 M), and the solution (50 mL) was adjusted to pH 11. After the reactor was placed in an ice bath at 0 °C with magnetic stirring for 1.0 h, 3-glycidoxypropyltrimethoxysilane (GLYMO, 1.5 g) was added dropwise under magnetic stirring within 0.5 h. Then, the mixture was heated to 65 °C for 6 h reaction, and then the reaction system was cooled to 0 °C in ice bath. In order to ensure the reaction between IDA and GLYMO and avoid the crosslinking reaction between GLYMO molecules, the above procedure was repeated twice including addition of GLYMO, reaction at 65 °C for 6 h, and cool-down in an ice bath at 0 °C. Finally, the obtained IDA-derived silane coupling agent solution was adjusted to pH 6 with concentrated HCl for the next grafting reaction on the surface of MS micropsheres. About 3 mg of prepared MS microspheres was transferred to a 1.5 mL Eppendorf tube and resuspended in 200 µL of PBS (pH 8.0). The MS microspheres were retained by a magnet, and

the supernatant was removed. This procedure was repeated three times. The MS microspheres were then incubated with 0.5 M Cu2+ aqueous solution and allowed to react at room temperature for 1 h. The MS microspheres were then retained by a magnet and the supernatant was removed, followed by 4× washing each in 200 µL of 20 mM NH4HCO3 (pH 8.0). For enzyme immobilization, the metal-ion chelated MS microspheres were incubated with 200 µL of TPCK treatedtrypsin (2 mg/mL) for 2 h under gentle rotation. After removal of the trypsin solution, the MS microspheres were washed four times with 200 µL of 20 mM NH4HCO3. The final product was stored in 20 mM NH4HCO3 and 0.02% sodium azide at 4 °C before use. The whole procedure for immobilization of trypsin onto the ion-chelated MS microspheres is shown in Figure 1. Preparation of On-Chip Enzymatic Reactor. The trypsinimmobilized MS microspheres were locally packed into the mirochannel by the application of a strong magnetic field using a magnet. A uniform suspension of MS microspheres with concentration 3 mg/mL was injected into the channel for 1 min, leading to a packed bed of ∼2-3 mm (Figure 2). To ensure a uniform suspension, the MS microspheres were agitated by autopipette mixing directly in the inlet vial immediately prior to the injection. A newly packed bed was used in each experiment. The 20 mM NH4HCO3 buffer solution (pH 8.0) was then flowed through the channel for 5 min. Characterization of Protein Immobilization on Metal-Ion Chelated MS Microspheres. A transmission electron microscope (JEM-2100F, JEOL, Tokyo, Japan) was used to characterize the morphology of the metal-ion chelated MS microspheres. To study the stability of the enzyme immobilization on MS microspheres, capillary electrophoresis/laser-induced fluorescence (CE-LIF) detection was explored. A homemade LIF detector (laser excitation wavelength: 473 nm; emission wavelength: 532 nm) was used for the detection. The chip was placed on an x-y-z translation stage. Any point of the channel Journal of Proteome Research • Vol. 6, No. 6, 2007 2369

technical notes


Figure 3. TEM images of (a) magnetic microspheres and (b) magnetic silica microspheres

could be selected and thus acted as the detection point optionally through adjusting the x-y-z stage. The fluorescence signals from the detection point were monitored continuously while a 20 mM NH4HCO3 buffer solution (pH 8.0) was used as the running solution propelled by electroosmotic flow with 900 V high voltage applied. On-Chip Tryptic Digestion and Identification. Two standard proteins, cytochrome c and bovine serum albumin, in 20 mM NH4HCO3 buffer solution (pH 8.0), were injected into the microchannel and digested with the prepared enzymatic reactor for 5 min at 50 °C. The resulting peptide fragments were then pumped out of the channel and collected in the waste reservoir with a pipet (2-20 µL, Eppendorf Research) for MALDI-TOF/TOF MS identification. For comparison, the digestions of Cyt-C and BSA were also performed by free trypsin in solution according to the conventional procedure (trypsin/ protein mass ratio 1:40, digestion time 12 h) and by the metalion chelated microreactor without trypsin immobilized, respectively. Extraction of Rat Liver. The rat liver tissue was cleaned with Milli-Q water to remove some possible contaminants, cut into small pieces, and homogenized in water containing 9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF) using a glass vessel in an ice bath. The resulting homogenate was swirled for 30 min and centrifuged for 20 min at 18000g. The supernatant was collected, fractionated in aliquots, and stored at -80 °C till further analysis. Protein concentration was measured using the Bradford assay using BSA as standard, 10 µg/µL for rat liver tissue. RPLC Separation of Rat Liver Extract. Agilent 1100 series capillary pumping system (DE, Germany) was used for gradient elution of samples. The capillary column of 30 cm length (250 µm i.d.) used in capillary RPLC was packed in house with 5 µm Zorbax 300SB-C8 particles. On-column frits were prepared by sol-gel method.41 A slurry packing procedure was employed. Mobile phase A consisted of 0.1% TFA in water and mobile phase B consisted of 0.1% TFA in acetonitrile (all vol/vol). The flow rate was 2 µL/min. Following a 10 min elution with 0% B, the separation was performed using gradient conditions as follows: 0% B increased up to 20% B in 30 min, then linearly increased to 90% B in 110 min, maintained at 90% B for 10 min for washing the column, and then ramped down to 0% B for equilibrium. Effluents were started to collect after 25 min 2370


Figure 4. Fluorescence intensity from the FITC-BSA immobilized microchannel versus electrophoresis time (excited at 473 nm and recorded at 520 nm).

gradient elution, and 70 RPLC fractions were collected every 1 min (2 µL) continuously regardless of the eluting profile shape. On column UV detection for RPLC was carried out at 215 nm using a Waters 484 tunable absorbance detector (Waters, MA) with modification, and then all fractions dried quickly at the room temperature. Afterward, the dried fractions were reconstituted in 2 µL of NH4HCO3 buffer solution (pH 8.0). Next step, each fraction was injected into on-chip microreactor to carry on tryptic digestion. The resulting peptide fragments were then pumped out of the channel and collected in the waste reservoir with a pipet (2-20 µL, Eppendorf Research) for MALDI-TOF/ TOF MS identification. Protein Identification Using MALDI MS. Digests of proteins (∼0.5 µL) were mixed with cyano-4-hydroxycinnamic acid (CHCA) matrix solution (in 50% acetonitrile and 0.1% TFA aqueous solution, v/v) at a 1:1 ratio on the MALDI plate. MALDI-TOF MS and MS/MS experiments were performed in positive ion mode on a 4700 proteomics analyzer (Applied Biosystems, Foster City, CA) with the Nd:YAG laser at 355 nm, a repetition rate of 200 Hz, and an acceleration voltage of 20 kV. For peptide mass fingerprinting (PMF) data, 800 laser shots were accumulated for each spectrum obtained from the TOF/ TOF. The TOF/TOF tandem mass spectra were acquired by the

technical notes

Li et al.

Figure 5. Effect of (A) incubation temperature, incubated for 5 min, and (B) incubation time, incubated at 50 °C, on sequence coverage of Cyt-C digests resulted from the digestion of MS microspheres packed on-chip microreactor.

data-dependent acquisition method with five precursor ions selected from one MS scan. Precursor selection was based on parent ions intensity. Data from the TOF/TOF were searched using Mascot (Matrix Sciences, London, U.K.) as the search engine. All searches of model proteins were performed against the Swiss-Prot database. The identifications of proteins from rat liver tissue were preformed against the NCBI database. GPS Explorer (Applied Biosystems) was used for submitting data acquired from TOF/TOF for database searching. The mass calibration was done externally on the target using a myoglobin digest peptide.

Results and Discussion Preparation of Packing Bed with Metal-Ion Chelated MS Microspheres inside the Microchannel. With the combination of the high surface area and orthogonal manipulations of smalldiameter MS microspheres, the fine fluidic controls of microdevices can result in a fast, sensitive, and reliable enzymatic methodology. As a building block for the system design, MS microspheres are a useful tool. Microsphere beds can be packed, dynamically positioned, flushed, and repacked in microchannels by magnetic field manipulations.12,14 This avoids some fabrication issues associated with other solid supports. In a flowing stream, a densely packed bed of small particles can be formed (Figure 2) which, in our study, was usually 2-3 mm in length. The bed region can be reproducibly formed, and once formed, resists deformation throughout a particular experiment. Once the experiment is finished, the MS microspheres could be flushed out quickly by removing the magnetic field and inducing flow. The packed MS microsphere bed is normally 2-3 mm in length. This is because if more MS microspheres were packed, the backpressure from the packed bed would be quite large. The forces from the backpressure would overwhelm the axial magnetic field gradients, and the bed would be flushed away.12 Characterization of Protein Immobilization on Metal-Ion Chelated MS Microspheres inside the Microchannel. Figure 3 shows the transmission electron microscope images of the morphologies of the metal-ion chelated MS microspheres. The uniform and small sizes of the microspheres (∼200 nm) provide a high surface area-to-volume ratio (S/V), which not only reduce diffusion distance (and time) for all steps in the procedure but also increase the density of binding sites for immobilization of enzyme within a given volume. Fluorescein isothiocyanate (FITC) conjugated BSA was adopted as the tracing mark by being immobilized on the MS microspheres. Before being collected near a magnet to form a packing bed within a microchannel, the MS microspheres were

washed by water and 20 mM NH4HCO3 buffer solution (pH 8.0) three times respectively to remove protein molecules either excessive or adsorptive nonspecifically. The stability of proteins immobilized on metal-ion chelated MS microspheres was investigated by CE-LIF detection. With respect that the fluorescence intensity represented the relative quantity of the immobilized fluorescent biomolecules, the fluorescence signals at one detection point were monitored while NH4HCO3 buffer solution (pH 8.0) flowed through the microchannel by propelling of the electroosmotic flow. The recorded fluorescence intensity was examined as a function of migration time (Figure 4), and the result showed that the fluorescence intensity was stable and no obvious decrease appeared during the 20 min process period. On-Chip Enzymatic Microreactor Application in MALDI Mass Mapping. Two large proteins with multiple cleavage sites, Cyt-C (MW ) 12 384) and BSA (MW ) 66 000), were used as model substrates for the proteolytic digestion. For comparison, the digestions of Cyt-C and BSA were also performed by free trypsin in solution for 12 h and by MS microspheres without immobilized trypsin, respectively. The proteolytic products were collected and analyzed by MALDI-TOF/TOF MS for identification of digested fragments. The standard temperature for tryptic digestion is 37 °C. Nevertheless, high incubation temperature has the effect of increasing flexibility and, therefore, the proteolytic susceptibility of a protein,42 which may lead to higher proteolytic efficiency. Proteins digestions were performed at different temperatures to investigate the influence of temperature on protein digestion efficiency. As displayed in Figure 5A, the sequence coverage of Cyt-C increased slowly at temperature lower than 50 °C, and decreased slightly at temperature higher than 50 °C. It is concluded that the modified trypsin exhibits optimal activity at 50 °C. Therefore, the following digestions with on-chip microreactor were all conducted at 50 °C. Figure 5B demonstrates the sequence coverage obtained from MALDI-TOF MS analysis for digests of Cyt-C by the onchip enzymatic microreactor with different incubation time. When the incubation time increased from 1 to 5 min, the sequence coverage of Cyt-C accordingly increased from 43% to 77%. With further increase of the incubation time, no significant change on sequence coverage was observed. Thus, the following experiments in this study were performed with an incubation time of 5 min. Figure 6 (1A and 2A) show the peptide mapping analysis of Cyt-C and BSA from the as-prepared on-chip microreactors. The samples were digested and positively identified. Many digest fragments were observed from the MS spectra. However, Journal of Proteome Research • Vol. 6, No. 6, 2007 2371


technical notes

Figure 6. MALDI-TOF mass spectrum of digests of (1) Cyt-C and (2) BSA using the MS microspheres packed on-chip microreactor (A) with immobilized trypsin and (B) without immobilized trypsin. Proteins are 0.20 mg/mL in 20 mmol/L NH4HCO3 buffer solution (pH 8.0). Proteins were all digested at 50 °C for 5 min. Eluent/matrix ratio is 1:1. Reflector mode was used to detect low-mass peptides. 2372


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Table 1. MALDI-TOF/TOF MS Results of Digestion Products by Metal-Ion Immobilized Trypsin On-Chip Microreactorsa Cyt-C protein

amino acids identified sequence coverage (%) digestion time peptides matched accession no. protein MW a

BSA

microreactor

in-solution

microreactor

in-solution

81

80

131

253

77

76

21

41

5 min 13

12 h 14

5 min 13

12 h 24

P00004 11694.1

P00004 11694.1

P02769 69248.4

P02769 69248.4

Three spot replicates were taken in the experiments.

Table 2. Detail-Identified Fragments of Cyt-C and BSA by MALDI-TOF/TOF MS Cyt-C

BSA

9-22 26-38 28-38 28-39 39-53 40-53 40-55 56-72 61-72

IFVQKCAQCHTVEK HKTGPNLHGLFGR TGPNLHGLFGR TGPNLHGLFGRK KTGQAPGFTYTDANK TGQAPGFTYTDANK TGQAPGFTYTDANKNK GITWKEETLMEYLENPK EETLMEYLENPK

25-34 35-44 161-167 233-241 242-248 347-359 360-371 402-412 413-433

61-73 80-86 80-87 89-99

EETLMEYLENPKK MIFAGIK MIFAGIKK TEREDLIAYLK

421-433 436-451 437-451 508-523

DTHKSEIAHR FKDLGEEHFK YLYEIAR ALKAWSVAR LSQKFPK DAFLGSFLYEYSR RHPEYAVSVLLR HLVDEPQNLIK QLINVCRDQFEKLGEYGFQNA LGEYGFQNALIVR VPQVSTPTLVEVSR KVPQVSTPTLVEVSR RPCFSALTPDETYVPK

the results of the compared experiment without immobilized trypsin showed that the protein molecules were not digested, because only numbers of peaks from the assisted matrix appeared in the low m/z range in Figure 6 (1B and 2B). A highly proteolytic efficiency has been confirmed for the proposed enzymatic microreactor in this work. Tables 1 and 2 list in detail the proteolytic results. The observation corresponded to the detection of fragments containing 81 out of the 104 possible amino acids of Cyt-C and 131 out of the 583 possible amino acids of BSA. The sequence coverage of 77% for Cyt-C and 21% for BSA from the database were obtained. The identification results could be compared with those by insolution digestion that required a reaction time of 12 h. Meanwhile, the sample volume is only 0.5 µL per analysis. Since the MS microspheres have a large surface area and the onchip microreactor has a very narrow space, the high concentration of immobilized enzyme would be achieved at a certain volume. Thus, the substrate might be in contact with trypsin for longer time if compared with the soluble system.43 Due to the high surface area-to-volume ratio of the MS microspheres packed within the microchannel, it is likely that there is a short distance for the substrate to diffuse from solution to the microstructured particles where trypsin was located. To further test the stability and reproducibility, six consecutive operations for Cyt-C with incubation of 5 min at 50 °C using the same on-chip MS microspheres packing bed were conducted, and resultant products were then analyzed by MALDI-TOF MS (Figure 7). Between each operation, the onchip microreactor was rinsed with 20 mM NH4HCO3 (pH 8.0) for 5 min. As demonstrated in Figure 7, for the first four runs,

Figure 7. Stability test of metal-ion immobilized trypsin on-chip microreactor.

the sequence coverage maintains at around 76% with relative standard deviation (RSD) of 1.7%. At the fifth run, the sequence coverage decreased to 59%, which indicates the beginning of the loss of the enzyme activity. Regeneration of the On-Chip Enzymatic Microreactor. Since our microreactor is prepared by packing the microchannel with MS microspheres which immobilized enzyme by CuIDA chelated adsorption, the major advantage of our on-chip microreactor over the covalently immobilized enzymatic microreactor is that the enzyme can easily be regenerated. According to Zou’s work40 and the stability test we conducted above, the prepared microreactor should be regenerated after four times of usage for digestion of proteins. The regeneration process includes the following steps: flushing the MS microspheres out of the microchannel by removing the magnetic field and inducing flow; elution of the metal-ion and enzyme from the particles with EDTA solution and then rechelating of the metal-ion onto the surface of the MS microspheres following the immobilization of the enzyme; and repacking the MS microspheres inside the microchannel in a flowing stream. The regeneration ability is evaluated with the digestion of Cyt-C under the same conditions before and after the regeneration of the on-chip microreactor. The digested products are analyzed with MALDI-TOF MS. Table 3 shows the MALDI-TOF MS results obtained from digestion products of 0.2 mg/mL Cyt-C by the on-chip microreactor before and after regeneration. It can be seen that the identified fragments are almost the same, which indicates a good reproducibility of the microreactor after regeneration process. Although in current work only trypsin immobilized microreactor has been exemplified, our on-chip microreactor is convenient to change the types of the immobilized enzymes, just by applying different enzyme solution as the same procedures for the regeneration process. Since each type of enzyme has the specificity for hydrolysis of proteins, enzymatic cleavage using different enzymes could give complementary structure information of a protein and increase the informative content of the map which consequently resolve ambiguous identifications.2,44 Digestion of Rat Liver Extract. In recent years, developments within column-based separations of proteins in proteomics have offered an optional strategy to analyze complex protein Journal of Proteome Research • Vol. 6, No. 6, 2007 2373

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Table 3. Summary of MALDI-TOF Results Obtained from Digestion Products of Cyt-C by Metal-Ion Immobilized Trypsin Microreactors Before and After Regenerationa before

amino acids identified sequence coverage (%) digestion time (min) peptides matched identified fragments with microreactors

a

after

81 77 5 13

80 76 5 12

9-22 IFVQKCAQCHTVEK 26-38 HKTGPNLHGLFGR 28-38 TGPNLHGLFGR 28-39 TGPNLHGLFGRK 39-53 KTGQAPGFTYTDANK 40-53 TGQAPGFTYTDANK 40-55 TGQAPGFTYTDANKNK 56-72 GITWKEETLMEYLENPK 61-72 EETLMEYLENPK 61-73 EETLMEYLENPKK 80-86 MIFAGIK 80-87 MIFAGIKK 89-99 TEREDLIAYLK

9-22 IFVQKCAQCHTVEK 26-38 HKTGPNLHGLFGR 28-38 TGPNLHGLFGR 28-39 TGPNLHGLFGRK 39-53 KTGQAPGFTYTDANK 40-53 TGQAPGFTYTDANK 40-55 TGQAPGFTYTDANKNK 56-72 GITWKEETLMEYLENPK 61-72 EETLMEYLENPK 61-73 EETLMEYLENPKK 80-86 MIFAGIK 89-99 TEREDLIAYLK

Three spot replicates were taken in the experiments.

Table 4. Proteins Identified from the RPLC Fraction 48# with On-Chip Tryptic Digestion Followed by MALDI-TOF MS/MS protein name

accession no.

protein MW

protein pI

pep. count

protein score

3-hydroxy-3-methylglutaryl-coenzy me A synthase 2 [Rattus norvegicus] TH2A histone [R. norvegicus] 3-hydroxyisobutyrate dehydrogenase [R. norvegicus] sterol carrier proteinsrat (fragment) fatty acid binding protein 1 [R. norvegicus] gametogenetin-binding protein 1 [R. norvegicus] R-globin [Rattus sp.]

gi|54035469 gi|57354 gi|83977457 gi|2119443 gi|56541250 gi|46410145 gi|30027750

56849.6 14275 35279.6 10462.5 14263.3 40828.3 9345.7

8.86 11.02 8.73 8.03 7.79 5.78 6.49

3 4 3 1 3 7 2

147 114 112 97 86 65 64

mixtures.45,46 The top-down methods provide purified protein for subsequent identification steps, but measurements designed to quantitate protein-level physicochemical properties (e.g., molecular weight, pI, hydrophobicity) have limited identification power. Thus, top-down methods should encompass protein fragmentation in order to generate sequence information and ultimately identification. Our replaceable trypsin immobilized on-chip microreactor provides a facile and lowcost way to solve this problem. Here, to demonstrate the applicability of the on-chip microreactor in top-down proteome analysis, it was applied to RPLC fraction of rat liver extract followed by analysis of the

Figure 8. RPLC chromatogram of the rat liver extract. One fraction is indicated with an arrow at 67-68 min (48#). See the Experimental Section for details of separation conditions. 2374


resulted digest by MALDI-TOF MS/MS. The RPLC chromatogram of the rat liver extract (20 µg) is shown in Figure 8. The RPLC fractions were collected in 1 min intervals from 20 to 110 min during the run. Each fraction was reconstituted by 2 µL of NH4HCO3 buffer solution (pH 8.0), and then pumped into the on-chip microreactor for digestion for 5 min at 50 °C. The resulted digests were then collected and analyzed by MALDITOF MS/MS. The RPLC fraction collected from 67 to 68 min (48#, indicated with arrow in Figure 8) was analyzed as a model. Numbers of peaks were observed from the mass spectrum of the resulted digests (data not shown), which indicates that the protein mixture was efficiently digested in the on-chip microreactor. The searches were performed against the NCBI database using combined PMF and MS/MS method, allowing for one missed cleavage. Peptide mass spectra were collected automatically with an acquisition mass range of 700-4000 m/z. External standards were used to achieve a mass accuracy of 100 ppm. Table 4 lists the proteins identified from the RPLC fraction 48#. According to the results, seven unique proteins with protein score more than 59 were identified. Since rat liver extract is highly complex protein sample, only one dimension of RPLC separation may not meet the need for complete fractionation. This leads to a large number of peptides after proteolytic digestion which is disbenefit for protein identification. Coupling digestion of RPLC fractions by on-chip microreactor with separation of digest by HPLC or CE will result in better identification of proteins, and this work is under investigation in our group.

Conclusions A replaceable and regenerable tryptic microreactor was prepared on chip by packing metal-ion chelated MS micro-

technical notes spheres within the microchannel. The microreactor was successfully applied to digestion of proteins. Similar identification results were obtained with much shorter digestion time on the microreactor compared to in solution digestion. The main advantage of the prepared microreactor with this approach is that the MS microspheres (which are retained by a magnet) can be fast and efficiently removed, which make the microreactor replaceable. Also, the MS microspheres can be easily regenerated, and good reproducibility before and after regeneration was obtained. Another advantage of our microreactor is that it can be constructed by immobilizing different types of enzyme for the peptide mapping analysis, which can provide complementary structure information of a protein. The results presented here clearly demonstrate that the prepared microreactor has shown great advantages in protein digestion, which might be of great significance in proteomic study. The efficiency of the digestion was further demonstrated by digestion of RPLC fractions of a real proteome sample, the rat liver extract. After the digestion of one RPLC fraction with on-chip microreactor followed by MALDI-TOF MS/MS analysis, a number of peaks were observed in the mass spectrum and 23 unique peptides corresponding to 7 proteins were identified.

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PR060558R

Journal of Proteome Research • Vol. 6, No. 6, 2007 2375

Microchip Reactor Packed with Metal-Ion Chelated Magnetic Silica

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