Rapid Characterization of Protein Chips Using Microwave-Assisted

ARTICLE pubs.acs.org/Langmuir

Rapid Characterization of Protein Chips Using Microwave-Assisted Protein Tryptic Digestion and MALDI Mass Spectrometry Na Young Ha, Shin Hye Kim, Tae Geol Lee, and Sang Yun Han* Center for Nano-Bio Convergence, Korea Research Institute of Standards and Science, Daejeon 305-340, Republic of Korea ABSTRACT: We demonstrate that the microwave-assisted protein enzymatic digestion (MAPED) method can be successfully applied to the mass spectrometric characterization of proteins captured on the affinity surfaces of protein chips. The microwave-assisted on-chip tryptic digestion method was developed using a domestic microwave, completing the on-chip proteolysis reaction in minutes, whereas the previous on-chip digestion methods by incubation took hours of incubation time. For the model protein chips, antibody-presenting surfaces were prepared, where anti-R-tubulin1 and antibovine serum albumin (BSA) were immobilized on self-assembled monolayers. The resulting digestion efficiency, displaying sequence coverages of 30 and 14% for R-tubulin1 and BSA, respectively, was comparable to the previous time-consuming incubation studies. It allowed the characterization of immunosensed proteins by MASCOT search using peptide mass fingerprinting. In an example of this method for protein chip applications, BSA naturally involved in fetal bovine serum was unambiguously identified on a model protein chip by imaging mass spectrometry. This work shows that biomass spectrometry techniques can be implemented for surface mass spectrometry and biochip applications. Along with recent advances in imaging mass spectrometry, this technique will provide a new opportunity for high-speed, and thus high-throughput in the future, label-free mass spectrometric assays using protein arrays.

1. INTRODUCTION MALDI (matrix-assisted laser desorption/ionization) mass spectrometry has provided a powerful means for the highthroughput mass spectrometric identification of biomolecules such as proteins, which has reinforced proteomics research together with ESI (electrospray ionization) mass spectrometry.1,2 In addition, by virtue of featuring the direct interrogation of sample surfaces, MALDI mass spectrometry has been recognized for further research opportunities such as imaging mass spectrometry3,4 and label-free assays using biochips.5
8 Imaging mass spectrometry localizes specific biomolecules such as lipids, peptides, and proteins on tissue samples, providing chemical mapping and facilitating a new method of biomarker discovery and disease diagnostics. By developing the on-tissue enzymatic digestion technique combined with imaging mass spectrometry, even simultaneous characterization and the localization of proteins on tissues become possible.9,10 Recently, new imaging time-of-flight mass spectrometers operated in novel microscope mode equipped with fast position-sensitive detectors are actively being developed and will soon bring high-throughput and high-resolution capability to imaging mass spectrometry.4,11 The application of MALDI mass spectrometry to biochips is another challenging subject for bioassays because it does not require samples to be labeled, allowing label-free and thus unbiased sensing and screening of biomolecular interactions.5
7 In applications, SAMs (self-assembled monolayers) offer versatile platforms for various biomimicking surfaces by providing appropriate r 2011 American Chemical Society

physicochemical surface properties and immobilizing biomolecules on the chip surfaces to probe specific biomolecular interactions.5,12,13 Often referred to as SAMDI (self-assembled monolayers for MALDI) mass spectrometry, alkanethiol SAMs on gold are known for their amenability to MALDI mass spectrometry.14
17 In typical SAMDI experiments, biochemical changes reflected in the observed molecular weights of biomolecules from the surfaces were primarily used to monitor enzymatic activities.18
20 In addition, monitoring protein
protein interaction was also demonstrated by observing intact ion peaks of proteins captured on biologically tailored SAM surfaces using MALDI mass spectrometry.21
23 However, the SAMDI method mainly relied on the measurement of the molecular weights of ion peaks observed directly from biologically tailored surfaces. In protein chip technology, microarrays of capture agents such as antibodies and aptamers serve a platform that detects proteins by specific interaction.6 As for detection methods, methods based on chemiluminescence or fluorescence in which the probes are tagged with enzymatic or fluorescent labels can be utilized. However, labeling proteins yields some disadvantages, for example, the nonspecificity of the protein
antibody interaction in an ELISA-based detection, the effects of fluorophore tags on molecular binding, and the like. For these reasons, the techniques of Received: May 13, 2011 Revised: July 5, 2011 Published: July 20, 2011 10098

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Figure 1. Schematic of microwave-assisted on-chip tryptic digestion for the mass spectrometric characterization of protein chips.

label-free detection, such as mass spectrometry and imaging SPR (surface plasmon resonance)24 or a combined approach of the two methods,25
27 have been an active subject of recent development, but they still need further development. For protein chips, in order to exploit the unique advantage that the mass spectrometric detection may provide, the characterization of proteins sensed on the affinity surfaces is essential but requires a further sample preparation step, namely, the proteolysis step facilitating protein identification using a technique such as peptide mass fingerprinting (PMF).28,29 In this regard, a few efforts to develop on-chip (i.e., on-surface) proteolysis methods have been reported. The previous reports examined the on-chip enzymatic digestion of proteins, which were physically adsorbed on the hydrophobic or hydrophilic chromatographic surfaces30,31 for SELDI (surface-enhanced laser desorption/ionization)32 or were immunosensed on the antibody-presenting chips by protein interactions.27,33 All of the previous developments were based on the conventional incubation method, where the incubation of a few hours for enzymatic cleavage was employed.27,30,31,33 The studies produced 5
15 on-chip tryptic peptides of proteins, which account for a sequence coverage of 15
40% for given proteins. Although the numbers of identified tryptic peptides did not seem to be large enough, they were still found to be sufficient for characterizing the proteins retained on the affinity surfaces by PMF. However, the previous incubation methods required a few hours for enzymatic reaction, creating a disadvantage for the practical use of mass spectrometric characterization because one of the main goals pursued by biochips is high-speed screening. In an effort to address the issue of the time-consuming process, we explored the possible application of microwave technology to protein chip assays (Figure 1). During the past few decades, microwave technology has been established as a promising opportunity to address the issue of time-consuming sample preparation in many chemical and biological applications. The technology has been demonstrated for its catalytic effects in increasing the reaction yields and reducing the incubation time for enzymatic reactions, which has become a standard step in many protocols of biological sample preparations.34 The mechanism of the catalytic effects from microwave irradiation is still being debated, and it may differ by application. However, previous studies suggested that the effects of microwave irradiation might not be solely thermal; rather, a certain agitation caused by microwave irradiation in molecular structures and motions might be involved in the catalytic effects of this technique as well. In the field of mass spectrometry-based proteomics in particular, the microwave-assisted protein enzymatic digestion (MAPED) method has been widely adapted to benefit large-scale highthroughput analyses of proteins utilizing liquid chromatography and mass spectrometry.35,36 Many further studies are still being carried out to develop ultrafast and sensitive methods combining

microwave irradiation with various techniques, examples of which include using enzyme-immobilized beads and magnetite beads in lab on a chip.37
39 However, the applicability of microwave technology to mass spectrometric characterization of biologically tailored surfaces has never been investigated to date. In this article, we report that microwave-assisted enzymatic digestion is applicable to the rapid characterization of protein chips using MALDI mass spectrometry by demonstrating efficient on-chip tryptic digestion in minutes, which allows the identification of surface proteins by PMF.

2. MATERIALS AND METHODS 2.1. Reagents and Materials. The gold-plated silicon wafer used as the SAM substrate was obtained from K-MAC (Daejeon, Korea). The linker reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and the PBS buffer were purchased from Thermo Fisher Scientific. The thiol reagent with terminal
COOH (HS-(CH2)11-(OCH2CH2)6-OCH2COOH) used for the preparation of SAMs on gold was purchased from Nanoscience Instruments, Inc. The thiol reagent with terminal
OH (HS-(CH2)11-(OCH2CH2)3-OH), R-cyano-4-hydroxycinnamic acid (CHCA), 20 ,40 ,60 -trihydroxyacetophenone (THAP), and other chemical reagents including ammonium bicarbonate, sodium bicarbonate, ethanol amine, trifluoroacetic acid (TFA), methanol, ethanol (HPLC-grade) were obtained from Sigma-Aldrich. As for proteins and antibodies, bovine serum albumin (BSA) and anti-BSA were obtained from Sigma-Aldrich, and R-tubulin1 (TUBA1, TUBA4A) and anti-R-tubulin1 were obtained from Calbiochem. Antitransferrin was obtained from DACO. Fetal bovine serum (FBS) was obtained from PPA. The trypsin gold from Promega was used for proteolysis. All of the reagents were used as received without further purification. 2.2. Preparation of Amine-Reactive Mixed SAMs on Gold. The gold-plated silicon wafer was cleaned using a superpiranha solution (H2O2, HNO3, and H2SO4 in a 10:1:6 volume ratio). The wafer was thoroughly rinsed with deionized water and allowed to dry under ambient conditions and was then cut into ∼1  1 cm2 chips. On the cleaned gold surfaces, the mixed SAMs exposing
OH and
COOH functional groups on the SAM surfaces were grown by immersing the chips in the ethanol solution of thiol reagents with
OH and
COOH terminals mixed in a 9:1 volume ratio (total 1 mM), typically for 15 h. The chips were taken out and washed with 50% ethanol and dried under a gentle stream of nitrogen. The molecular composition of the mixed SAMs was routinely monitored by the SAMDI method. In the mixed SAMs, the COOH functional groups served as a reaction site for further antibody immobilization by EDC/sulfo-NHS chemistry, and the poly(ethylene glycol) moiety reduced the nonspecific adsorption of unwanted proteins on the SAM surfaces. Immediately after the washing step, the surface was activated with amine-reactive NHS esters by treatment with a solution of 75 mM EDC/15 mM sulfo-NHS for 30 min. Then, the chips were dried under ambient conditions or in a vacuum. 10099

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2.3. Immobilization of Antibodies on Amine-Reactive Surfaces. The antibody molecules were immobilized on the activated surface by forming a covalent amide bond with the amine-reactive surface. Typically, 10 μL of antibody solution (1 μg/μL) in 50 mM sodium bicarbonate buffer (pH 9.2) was applied to the activated surface for 90 min at room temperature. During the reaction time, the chips were covered with a plastic lid to prevent the solution from drying. After the immobilization step, the chips were washed with a PBS buffer at least three times to remove excess antibody molecules from the surfaces. The unreacted amine-reactive surface groups were then blocked using 0.5 M ethanolamine (pH 8.0) for 1 h, and the chips were washed twice with a PBS buffer and dried at room temperature. In the immunosensing experiments, a 10 or 15 μL drop of protein (antigen) solution (typically, 1 μg/μL in a PBS buffer) or diluted FBS was directly applied to the antibody-presenting surfaces. To remove proteins unbound or adsorbed by nonspecific interaction on the chip, the chips were thoroughly washed using a PBS buffer three times and 25 mM ammonium bicarbonate buffer (pH 7.8) and dried at room temperature.

2.4. On-Chip Tryptic Digestion by Incubation or Microwave Irradiation. Proteins, either captured antigens or immobilized antibodies, were subjected to on-chip tryptic digestion for subsequent mass spectrometric characterization. On-chip tryptic digestion was performed by applying a 10 or 15 μL drop of trypsin solution in an ammonium bicarbonate buffer (pH 7.8). For the incubation method, on-chip tryptic digestion was carried out in an incubator set at 37 °C, the inside of which was maintained to be humid so that the tryptic solution was not dried out during the cleavage reaction. After the incubation, the chip was dried in a vacuum desiccator. In microwave-assisted on-chip tryptic digestion, the same method as in the above incubation experiments was used in applying the trypsin solution to the chip. The main difference was that the digestion step was performed in an inexpensive domestic microwave (700 W, model KRB20YW, Daewoo Electronics, Korea) instead of an incubator. In this method, the chip was placed in a Petri dish, which was floated on water in a plastic beaker in order to dissipate the extra microwave energy.40 On-chip tryptic digestion was then typically carried out at a 250 W power level for 4 min.

2.5. MALDI and Imaging Mass Spectrometry of Tryptic Peptides on the Surfaces. In the SAMDI method used to monitor alkanethiol SAMs on gold, THAP (5 mg/mL in 50% MeOH) was employed as the matrix. For the MALDI mass spectrometry of tryptic peptides produced on the chip, CHCA was chosen as the matrix (5 mg/mL in 50% ACN with 0.5% TFA). After the on-chip digestion step, the matrix solution was directly applied to the chip. After it was dried, the chip was attached to a MALDI target plate using double-sided conductive tape and was subjected to mass analysis using a MALDI-TOF mass spectrometer (Autoflex III, Bruker Daltonics, Germany). MALDI mass spectra were taken for positive ions in reflectron mode, and a 355 nm laser operating at 200 Hz with a beam diameter of about 100 μm was employed as the ionization laser. Typically, 400 shots of laser pulses were used to obtain a single mass spectrum. The observed peaks that were due to trypsin autolysis provided a convenient way to make internal calibrations (m/z = 842.5, 1045.6, 2211.1). MALDI imaging mass spectra were taken in microprobe mode using a linear detector whose mass images were produced using flexImaging software (Bruker Daltonics). The spacing between two points was set at 400 μm, and it took about 20 min to take a mass image for a circled area with a diameter of 8 mm.

3. RESULTS AND DISCUSSION 3.1. Mass Spectrometry of Antibody-Presenting SAMs on Gold. In preparing model biosurfaces for protein chips, more

specifically, antibody-presenting chips, we utilized the surface

Figure 2. MALDI mass spectra of (a) the mixed SAMs of alkanethiol on gold, where M stands for the disulfide of HO-(CH2CH2O)3-(CH2)11-SS-(CH2)11-(OCH2CH2)3-OH and M0 stands for the mixed disulfide of HO-(CH2CH2O)3-(CH2)11-S-S-(CH2)11-(OCH2CH2)6-OCH2-COOH, and (b) the anti-BSA-presenting SAMs after on-chip tryptic digestion (37 °C, 3 h), where T denotes the peaks for trypsin autolysis and A is for tryptic peptides of immobilized antibody molecules.

chemistry of alkanethiol SAMs on gold. The surface chemistry is well established and has been extensively demonstrated to model various biomimicking systems,12,13 and the SAM surface itself is susceptible to MALDI mass analysis as well.14
17 However, it is to be noted that because the present study deals with proteins adsorbed on the affinity surfaces this study may not be restricted to the use of alkanethiol SAMs on gold as the biochip surfaces. Figure 2a displays the SAMDI mass spectrum for the mixed SAM surface generated using the thiol reagents mixed in a ratio of 9:1 (
OH thiol/
COOH thiol). The observed ions were mainly the disulfide species of HO-(CH2CH2O)3-(CH2)11-SS-(CH2)11-(OCH2CH2)3-OH (denoted by M) and HO-(CH2CH2O)3-(CH2)11-S-S-(CH2)11-(OCH2CH2)6-OCH2-COOH (M0 ), such as [M + Na]+and [M + K]+ as well as [M0 + Na]+, [M0 + K]+, and [M0
H + Na2]+. The disulfide species in the mass spectrum indicate that the SAMs were indeed well fabricated on the gold surfaces,17 and the ratio between the peak areas of M and M0 groups allowed us to monitor the relative abundance of the two functional groups (
OH and
COOH) on the surfaces. Thiol ratios of 2:1, 4:1, 9:1, and 18:1 were examined for their immunosensing capability. The 9:1 ratio was found to be the most efficient under our experimental conditions and was used throughout this work. Using the known linker chemistry (EDC/sulfo-NHS), antibody molecules were covalently bound to the SAM surfaces, providing model immunosensing surfaces for the present study. The prepared antibody-presenting surface was mass analyzed using on-chip tryptic digestion by incubating the enzymatic reaction at 37 °C for 3 h. The resulting mass spectrum of tryptic peptides arising from antibody molecules directly obtained from the chip surface is given in Figure 2b. In fact, irrespective of the identities of antibody molecules, the observed tryptic peptides from immobilized antibodies could be mostly explained by the expected digests for IgG as a result of the structural similarity of antibody molecules. This fact offers a useful tip for discriminating the contribution of antibody-presenting surfaces in the mass spectra of on-chip digested proteins. The peak lists for tryptic digests of IgG (denoted by A in the mass spectra), trypsin autolysis (T), 10100

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Table 1. Observed Tryptic Peptides of Antigen Proteins after On-Chip Digestion by Incubation (37 °C, 3 h) theoretical observed m/z

Figure 3. MALDI mass spectra after on-chip tryptic digestion (37 °C, 3 h) of (a) R-tubulin1//anti-R-tubulin1 and (b) BSA//anti-BSA, where peaks for tryptic peptides of R-tubulin1 (*), BSA (*), and antibody (A), trypsin autolysis (T), and background peaks from SAMs (() are identified.

along with typical contaminants frequently observed from our SAM surfaces were prepared and used in identifying background peaks. A control experiment was performed by passivating aminereactive esters using ethanolamine (pH 8.0) before antibody molecules were loaded. In the experiment, no significant peaks of tryptic peptides of antibody molecules were observed, ensuring that the antibody-presenting surface accommodates antibodies in a covalent way. 3.2. On-Chip Tryptic Digestion by Conventional Incubation (37 °C, 3 h). On-chip tryptic digestion using the conventional incubation method was examined for the immunosensing chips prepared in this work. In this work, a drop (10
15 μL) of trypsin solution (pH 7.8) was loaded onto the surface of the antibody-presenting chip, where antigen proteins were already captured by specific interactions with the affinity surface. The chips were then incubated at 37 °C for 3 h. The incubation conditions were carefully inspected with respect to time and trypsin concentration. It was found that an incubation time of at least 3 h was necessary to achieve discernible digestion. An incubation of longer than 3 h did not yield a significant improvement in the digestion efficiency. Figure 3 shows representative mass spectra for on-chip tryptic peptides produced on the model immunosensing systems: (a) R-tubulin1 captured on the anti-R-tubulin1-presenting surface (R-tubulin1//anti-R-tubulin1, denoted by the combination of antigen//antibody) and (b) BSA retained on the anti-BSA surface (BSA//anti-BSA). The results are summarized in Table 1. The MALDI mass spectrum for the on-chip tryptic digestion of R-tubulin1//anti-R-tubulin1 revealed 13 tryptic peptides of R-tubulin1 protein, which explains 36% of the sequence coverage. For the BSA//anti-BSA chip, eight tryptic peptides of BSA were observed, corresponding to a 16% sequence coverage. The peak assignments were aided by theoretically predicted lists of tryptic digests from the known protein sequences, where other possible factors such as post-translational modifications were not considered. Previously, Caputo et al. investigated the on-chip tryptic digestion of GCDFP-15/PIP protein captured on a chromatographic surface (H4, SELDI ProteinChip).30 After 2
4 h of incubation for the on-chip enzymatic cleavage, several tryptic

m/z

start


missed

end

cleavage

sequence

1715.9 2303.2

R-Tubulin1//anti-R-Tubulin1 1715.8 65
79 0 AVFVDLEPTVIDEIR 2303.1 65
84 1 AVFVDLEPTVIDEIRNGPYR

2415.2

2414.9 85
105

1

QLFHPEQLITGKEDAANNYAR

1023.5

1023.5 97
105

0

EDAANNYAR

1779.8

1778.8 97
112

1

EDAANNYARGHYTIGK

1826.0

1825.7 106
121

1

GHYTIGKEIIDPVLDR

2095.2

2095.1 106
123

2

GHYTIGKEIIDPVLDRIR

1338.8

1338.5 113
123

1

EIIDPVLDRIR

1718.9 1487.8

1718.8 216
229 1487.8 230
243

0 0

NLDIERPTYTNLNR LISQIVSSITASLR

2409.2

2409.1 244
264

0

FDGALNVDLTEFQTNLVPYPR

1757.0

1756.8 265
280

0

IHFPLATYAPVISAEK

2330.0

2330.0 403
422

0

AFVHWYVGEGMEEGEFSEAR

1468.8

1468.5 25
36

2

DTHKSEIAHRFK

977.5

977.3

123
130

0

NECFLSHK

BSA//anti-BSA

927.5

927.3

161
167

0

YLYEIAR

1517.9 1439.8

1517.8 236
248 1439.7 360
371

2 1

AWSVARLSQKFPK RHPEYAVSVLLR

1479.8

1479.7 421
433

0

LGEYGFQNALIVR

988.6

988.4

490
498

1

TPVSEKVTK

1504.9

1505.2 549
561

1

QTALVELLKHKPK

peptides of GCDFP-15/PIP protein were observed. Ge et al. demonstrated that a complete chemical and enzymatic treatment of proteins including reduction, alkylation, and enzymatic cleavage procedures could be performed on chromatographic chips (H4 and NP20, SELDI ProteinChip).31 Under the protocol, proteins underwent proteolysis, yielding 5
15 peptide digests accounting for 17
42% sequence coverage, where the step for enzymatic cleavage involved 2 h of incubation. Not only on the chromatographic SELDI surfaces were cases of on-chip digestion on immunosensing surfaces also known. Bellon et al. showed the on-chip mass spectrometric characterization of ovalbumin and β-lactoglobulin retained on the antibody-presenting SAM chips that were devised to be compatible with imaging SPR (SPRi) experiments.27 The incubation took 1 h at room temperature, and six and four tryptic peptides were observed on the chip for ovalbumin and β-lactoglobulin, which were 24 and 35% sequence coverages, respectively. Seok et al. also examined the onchip digestion protocol on the antibody-presenting immunosensing surfaces in some detail.33 Their protocol produced 10 tryptic peptides for BSA after 3 h of incubation (16% sequence coverage). Although the numbers and sequence coverages of the identified tryptic peptides in the previous studies were not high, they were still found to be suitable for characterizing proteins captured on the affinity surfaces in a MASCOT database search (www.matrixscience.com). The previous studies using the conventional incubation have demonstrated the application of onchip digestion to mass spectrometric detection for label-free protein chip assays. However, the requirement of a couple of hours for the incubation creates certain limitations on its application for 10101

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Figure 4. MALDI mass spectra after the on-chip tryptic digestion of BSA//anti-BSA with different microwave irradiation times of (a) 30 s, (b) 1 min, and (c) 4 min, where peaks for tryptic peptides of BSA (*) and antibody (A), trypsin autolysis (T), and background peaks from SAMs (() are identified.

high-speed, high-throughput assays using protein chips, which is one of the main goals that the biochip technology ultimately pursues. 3.3. Microwave-Assisted On-Chip Tryptic Digestion (250 W, 4 min). Using the same antigen
antibody sets examined for the above incubation method, we investigated the effect of microwave irradiation on the on-chip enzymatic cleavage reaction. Prior to the experiments, we examined the cleavage conditions with respect to microwave power (250, 500, and 700 W) and irradiation time (0.5, 1, 2, 3, and 4 min), where irradiation by a 250 W microwave for 4 min was found to be optimal for this work. Figure 4 demonstrates the effects of the microwave irradiation time. Even after a short irradiation of 30 s, a few tryptic peptides were already produced. The number of observed tryptic digests increased as the irradiation time increased, and the enzymatic digestion reaction became clearly pronounced after 3 min of microwave irradiation. The temperature of the water reservoir was about 60 °C, when measured immediately after the microwave exposure for 4 min. Irradiation for longer than 4 min did not improve the cleavage efficiency but caused only a significant evaporation of trypsin solution on the chip. For a comparison of the digestion efficiency, we also carried out the microwave-assisted tryptic digestion of proteins in solution. In the in-solution study, the mixture of protein and trypsin solutions, each prepared at the same concentrations used for the onchip experiments but with a larger volume of 50 μL, was subjected to microwave irradiation. After microwave irradiation for 4 min at 250 W, the solution was mass analyzed by MALDI mass spectrometry. In reality, a higher protein concentration was used for the in-solution study because only a tiny portion of proteins was captured and subjected to the on-chip experiments. Unlike the in-solution experiment, the on-chip digestion conducted in a small droplet (∼ 10 μL) of trypsin solution on the exposed chip surface has certain subtleties in control as well. The resulting mass spectra of in-solution and on-chip digestion with the assistance of microwave irradiation are given in Figures 5 and 6 for R-tubulin1 and BSA, respectively. The observed tryptic peptides under both conditions for the two protein cases are also compared in Tables 2 and 3. As shown in the mass

ARTICLE

Figure 5. MALDI mass spectra after microwave-assisted tryptic digestion of (a) R-tubulin1 in solution and (b) R-tubulin1//anti-R-tubulin1, where peaks for tryptic peptides of R-tubulin1 (*) and antibody (A), trypsin autolysis (T), and background peaks from SAMs (() are identified.

Figure 6. MALDI mass spectra after the microwave-assisted tryptic digestion of (a) BSA in solution and (b) BSA//anti-BSA, where peaks for tryptic peptides of BSA (*) and antibody (A), trypsin autolysis (T), and background peaks from SAMs (() are identified.

spectra given in Figures 5b and 6b, microwave irradiation caused meaningful enzymatic cleavages of proteins captured on the chips in a limited reaction time of 4 min. It yielded, typically, 14 tryptic peptides for R-tubulin1//anti-R-tubulin1 (30% sequence coverage), which is comparable to the microwave-assisted in-solution results showing 14 tryptic peptides (38%, Figure 5a) and also comparable to the on-chip results by the incubation of 13 tryptic digests (36%, Figure 3a). As for BSA//anti-BSA, 10 tryptic peptides (14% sequence coverage) were observed in the mass spectrum for microwave-assisted on-chip digestion whereas microwave-assisted in-solution digestion and on-chip digestion by incubation resulted in 16 (30%, Figure 6a) and 8 peptides (16%, Figure 3b), respectively. The observed sets of tryptic peptides after the microwave-assisted on-chip digestion were examined by a MASCOT search for their capability in characterizing proteins by PMF. With respect to the tryptic peptide sets, the MASOCT search (missed cleavage: 2) returned the exact protein identities of R-tubulin1 and BSA with respective 10102

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Table 2. Observed Tryptic Peptides of r-Tubulin1 Captured on the Antibody Surfaces (r-Tubulin1//anti-r-Tubulin1) after Microwave-Assisted In-Solution and On-Chip Digestion (250 W, 4 min) theoretical (m/z)

observed in solution (m/z)

observed on a chip (m/z)

start
end

missed cleavage

sequence

1715.9

1715.8

1715.8

65
79

0

AVFVDLEPTVIDEIR

2303.2 1410.8

2303.0 1410.7

2302.9

65
84 85
96

1 0

AVFVDLEPTVIDEIRNGPYR QLFHPEQLITGK

2415.2

2415.0

2414.9

85
105

1

QLFHPEQLITGKEDAANNYAR

1779.8

1778.8

1778.8

97
112

1

EDAANNYARGHYTIGK

1826.0

1825.8

1825.9

106
121

1

GHYTIGKEIIDPVLDR

2095.1

106
123

2

GHYTIGKEIIDPVLDRIR

1069.5

1069.6

113
121

0

EIIDPVLDR

1338.5

113
223

1

EIIDPVLDRIR

1718.7 1487.8

1718.8 1487.8

216
229 230
243

0 0

NLDIERPTYTNLNR LISQIVSSITASLR FDGALNVDLTEFQTNLVPYPR

2095.2 1069.6 1338.8 1718.9 1487.8 2409.2

2409.0

2409.2

244
264

0

1757.0

1756.8

1756.9

265
280

0

IHFPLATYAPVISAEK

1730.8

1730.5

312
326

1

YMACCLLYRGDVVPK

1807.9

1807.8

1380.7

1380.7

1536.8 2330.0

1536.7 2330.3

374
390

0

AVCMLSNTTAIAEAWAR

391
401

1

LDHKFDLMYAK

391
402

2

LDHKFDLMYAKR

403
422

0

AFVHWYVGEGMEEGEFSEAR

Table 3. Observed Tryptic Peptides of BSA Captured on the Antibody Surfaces (BSA//anti-BSA) after Microwave-Assisted In-Solution and On-Chip Digestion (250 W, 4 min) theoretical (m/z)

observed in solution (m/z)

observed on a chip (m/z) 1193.6

start
end

missed cleavage

sequence

1193.6

1193.5

25
34

1

DTHKSEIAHR

1249.6

1249.6

35
44

1

FKDLGEEHFK

2435.2 1163.6

2435.4 1163.6

45
65 66
75

0 0

GLVLIAFSQYLQQCPFDEHVK LVNELTEFAK

1349.5

1349.6

977.5 927.5

927.4

1083.6 2045.0

78
88

0

TCVADESHAGCEK

977.4

123
130

0

NECFLSHK

927.5

161
167

0

YLYEIAR

1083.5

161
168

1

YLYEIARR

168
183

1

RHPYFYAPELLYYANK

1001.6

233
241

1

ALKAWSVAR

1517.8 1177.6

236
248 300
309

2 0

AWSVARLSQKFPK ECCDKPLLEK

2044.9

1001.6 1517.9 1177.6

1517.7

2457.2

2457.1

341
360

2

NYQEAKDAFLGSFLYEYSRR

1567.7

1567.7

347
359

0

DAFLGSFLYEYSR

1439.8

1439.7

360
371

1

RHPEYAVSVLLR

1283.7

1283.6

361
371

0

HPEYAVSVLLR

1305.7

1305.7

402
412

0

HLVDEPQNLIK

1479.8

1479.8

1479.7

421
433

0

LGEYGFQNALIVR

1639.9 988.6

1639.9 988.5

437
451 490
498

1 1

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scores of 150 and 66, which was as successful as in the microwaveassisted in-solution cases with scores of 177 and 127, respectively. Our results clearly show that the microwave-assisted on-chip tryptic digestion technique can be applicable to the mass spectrometric detection or characterization of proteins in protein chip technology. The microwave technique is comparable in cleavage efficiency to the previous on-chip digestion method that took hours of incubation time while completing the enzymatic cleavage

reaction just in minutes. During the given time, it catalyzes the proteolysis reaction to a suitable level for protein characterization by PMF. We further examined other possibilities of cleavage pathways. We carried out an experiment by microwave irradiation of BSA// anti-BSA using the same buffer solution used for the trypsin solution simply by omitting trypsin. The effect of microwave-assisted on-chip acid hydrolysis was also examined. In the two studies, no 10103

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Figure 7. Mass images for tryptic peptides of BSA naturally involved in 20-fold-diluted FBS, captured on the anti-BSA and antitransfferin (antiTF) chip and produced by the microwave-assisted on-chip tryptic digestion method (250 W, 4 min).

proteolytic peptides were observed at all in subsequent investigation by MALDI mass spectrometry. This suggests that microwave irradiation indeed plays a role in catalyzing the enzymatic activity in the on-chip digestion reaction. The catalytic effect involved in this work may closely follow that of the in-solution MAPED reaction, although the different on-chip situation yielded a slightly different cleavage pattern from that of in-solution digestion (e.g., increase in missed cleavage). 3.4. Label-Free Immunoassay of BSA in FBS by Imaging Mass Spectrometry. As an example of immunosensing protein chip assays by label-free mass spectrometric detection, we demonstrated MALDI imaging mass spectrometry of BSA in fetal bovine serum (FBS) captured on the protein chip surface. For the demonstration, a simple chip presenting two antibodies, anti-BSA and antitransferrin, was prepared. In fact, because of the limited capability of the domestic microwave employed in this work, protein arrays with a larger size were not successful for microwaveassisted on-chip tryptic digestion. However, many microwave ovens with a large sample capacity and a uniform radiation of microwaves are commercially available, and they may be able to handle a larger format of protein arrays. The chip was immersed in 20-fold-diluted FBS and washed thoroughly with buffer solutions. It was then subjected to microwave-assisted on-chip tryptic digestion (250 W, 4 min). The resulting tryptic peptides of captured proteins were mass imaged by MALDI imaging mass spectrometry in the microprobe mode. From the set of mass spectra obtained from coordinates of the imaging area, mass peaks specific to tryptic peptides of BSA were chosen and visualized as mass images. The image for a given mass peak thereby carries information on the localization and quantity of the corresponding protein. Figure 7 displays the mass images obtained for tryptic peptides of BSA (m/z = 977.5, 988.6, 1352.7, and 1468.2). The images reveal that the BSA tryptic peptides are clearly detected on the anti-BSA spot. A small amount of BSA digests was also observed on the antitransferrin spot, which is probably due to nonspecific adsorption. The images for BSA digests are spatially well correlated, suggesting that the digests have a common origin. In fact, the serum contains a complex mixture of proteins, which brings out a complicated situation for biochip assays, including the nonspecific adsorption of proteins on the wrong antibody spot. However, by knowing that anti-BSA was prepared on the spot, one can look only at the mass images for the set of BSA

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tryptic peptides. As shown in Figure 7, the observed image set for BSA tryptic peptides ensures that the identified BSA proteins are indeed immunosensed on the chip. In addition, despite advance in modern surface engineering, biochip assays are still not completely free from nonspecific adsorption. This becomes more important when optical detection is used in the biochip assays. The nonspecific adsorption of BSA on the antitransferrin spot is identifiable in Figure 7. In other words, the nonspecific adsorption of other serum proteins occurring on the anti-BSA spot would never be detected by this mass spectrometric imaging method, altogether suggesting a scheme of interference-free detection. For this purpose, the implementation of on-chip enzymatic digestion becomes indispensible for these label-free mass spectrometric assays, where the microwave technique demonstrated in this work would play an important role by providing a fast and straightforward means of sample preparation. The present imaging mass spectrometry operated in microprobe mode requires a rather long time for image collection (e.g., 20 min per spot in this work). However, recent development in imaging mass spectrometers in microscope mode and fast position detectors, together with this fast sample preparation method, will soon offer a promising means for label-free high-throughput mass spectrometric assays using protein chips.

4. CONCLUSIONS This work reports a method for the rapid characterization of protein chips using microwave-assisted protein tryptic digestion and MALDI mass spectrometry. This method was shown to be well suited to protein arrays and imaging mass spectrometry, the combination of which will offer various strategies for the mass spectrometric analysis of protein chips. This simple, fast sample preparation method will greatly assist high-speed and later on high-throughput protein chip assays using MALDI mass spectrometry. ’ AUTHOR INFORMATION Corresponding Author

*Fax: +82-42-868-5032. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Biosignal Analysis Technology Innovation Program (2010-0020640) of MEST via KOSEF. ’ REFERENCES (1) Hillenkamp, F.; Peter-Katalinic, J. MALDI MS: A Practical Guide to Instrumentation, Methods, and Application; Wiley-VCH: Weinheim, Germany, 2007. (2) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (3) Cornett, D. S.; Reyzer, M. L.; Chaurand, P.; Caprioli, R. M. Nat. Methods 2007, 4, 828–833. (4) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26, 606–643. (5) Gurard-Levin, Z. A.; Mrksich, M. Annu. Rev. Anal. Chem. 2008, 767–800. (6) Mitchell, P. Nat. Biotechnol. 2002, 20, 225–229. (7) Lee, Y. S.; Mrksich, M. Trends Biotechnol. 2002, 20, S14–S18. (8) Evans-Nguyen, K. M.; Tao, S. C.; Zhu, H.; Cotter, R. Anal. Chem. 2008, 80, 1448–1458. (9) Groseclose, M. R.; Andersson, M.; Hardesty, W. M.; Caprioli, R. M. J. Mass. Spectrom. 2007, 42, 254–262. 10104

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