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A Radiate Microstructure MALDI Chip for Sample Concentration and Detection Shun-Yuan Chen,†,‡ Kun-In Li,† Chih-Sheng Yu,‡ Jun-Sheng Wang,‡ Yi-Chiuen Hu,*,‡ and Chien-Chen Lai*,†,§ Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, Instrument Technology Research Center, National Applied Research Laboratories, Taiwan, and Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan Although matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOFMS) analysis is an important tool for analyzing and characterizing biomolecules of varying complexity, the sensitivity of MALDITOFMS is dependent on proper preparation of the sample, a process that is oftentimes problematic and requires considerable expertise. In this study, we have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The sample/ matrix mixture was deposited onto the central space of the well on the chip and allowed to dry. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Optimizing processes of several preparation factors were also performed to ensure matrix homogeneity in our chip. Analysis of the samples with MALDI-TOFMS showed that the signals from samples on our chip were significantly greater than those on the unmodified plate. The feasibility of using this chip to detect peptides and phosphopeptides was also demonstrated. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a rapid, easy-to-use modality with a high sensitivity and widely detectable mass range for peptide mass fingerprinting.1 This technique relies on the utilization of an ultraviolet (UV) light absorbent matrix compound. The matrix and the sample are mixed in an appropriate proportion in solvent and deposited onto the sample plate for further MS analysis. However, there are still some problems in the application of this technique. One critical problem is obtaining sample/matrix cocrystalline structures for effective ionization. Analytes of MALDIMS are normally applied directly to sample plates, which often leads to the formation of irregular crystal spots. The uneven distribution of the spot can result in areas of analyte localization, or “sweet spots”. Although sample enrichment for trace analyte * Corresponding authors. (Y.-C.H.) Tel: (886) 3-57799911ext. 406. Fax: (886) 3-5773947. E-mail:
[email protected]. (C.-C.L.) Tel: (886) 4-22840485ext. 235. Fax: (886) 4-22858163. E-mail:
[email protected]. † National Chung Hsing University. ‡ National Applied Research Laboratories. § China Medical University. (1) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63 (1193), A-1203A. 10.1021/ac101426n 2010 American Chemical Society Published on Web 06/16/2010
might improve the performance of MALDI-MS,2 those procedural modifications do not minimize the time needed to search for sweet spots. Circumventing this limitation of MALDI analysis is crucial, especially for high throughput or automation. One feasible approach is to modify the surface of the MALDI target plate in a manner that would enrich the trace analytes before analysis. For example, chemical treatments that change the hydrophilic property of the metallic MALDI plate surface into one with hydrophobic characteristics have been used to concentrate and localize samples.3-6 A steep wettability gradient between the ultraphobic surrounding and the hydrophilic matrix provides optimal guidance for automated sample deposition.7 The hydrophilic surface anchors the deposited samples within the defined zone during solvent evaporation. Although these chemical methods are effective, the processes are somewhat complicated and may not be easy to reuse via standard rinse procedures. Single-use or disposable conductive layers, such as those found in polymeric chips8,9 and silicon chips,10 attached to the MALDI plate have been shown to improve the performance of MALDI analysis. Wei and Siuzdak found that porous silicon could replace the matrix, leading to improved performance in small-molecule analysis.11 Several modifications have also been made to the surface of the silicon substrate to allow the MALDI sample plate to be used for different applications.12-15 (2) Pan, C.; Xu, S.; Zhou, H.; Fu, Y.; Ye, M.; Zou, H. Anal. Bioanal. Chem. 2007, 387, 193–204. (3) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436–3442. (4) Konig, S.; Grote, J. Biotechniques 2002, 32 (4), 912, 914–915. (5) Redeby, T.; Roeraade, J.; Emmer, A. Rapid Commun. Mass Spectrom. 2004, 18, 1161–1166. (6) Gundry, R. L.; Edward, R.; Kole, T. P.; Sutton, C.; Cotter, R. J. Anal. Chem. 2005, 77, 6609–6617. (7) Poetsch, A.; Schlu ¨ sener, D.; Florizone, C.; Eltis, L.; Menzel, C.; Ro ¨gner, M.; Steinert, K.; Roth, U. J. Biomol. Technol. 2008, 19, 129–138. (8) Iba´n ˜ez, A. J.; Muck, A.; Svatos, A. J. Mass Spectrom. 2007, 42, 634–640. (9) Dunn, J. D.; Igrisan, E. A.; Palumbo, A. M.; Reid, G. E.; Bruening, M. L. Anal. Chem. 2008, 80, 5727–5735. (10) Zhou, H.; Xu, S.; Ye, M.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Han, G.; Fu, Y.; Zou, H. J. Proteome Res. 2006, 5, 2431–2437. (11) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243–246. (12) Hsieh, S.; Ku, H. Y.; Ke, Y. T.; Wu, H. F. J. Mass Spectrom. 2007, 42, 1628–1636. (13) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Int. J. Mass Spectrom. 2003, 226, 107–116. (14) Chen, Y. Q.; Bi, F.; Wang, S. Q.; Xiao, S. J.; Liu, J. N. J. Chromatogr., B 2008, 875, 502–508.
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Scheme 1. Fabrication of the Microstructure Chip (ICP: inductively coupled plasma, PR: photoresist, PPFC: plasma polymerization fluorocarbon)
Herein we demonstrate another method for MALDI sample enrichment and autodeposition with a microscale radiate silicon chip.16 We found that, upon application of the sample to the chip, the gradient generated by the hydrophobic microtexture silicon surface drove the sample to the center of the chip, resulting in a concentrated sample spot on the chip. Different sample/matrix proportions were tested to acquire the best crystalline-forming conditions. The results of the analysis, and the relative merits and pitfalls of these techniques, are discussed. MATERIALS AND METHODS Materials. The radiate microstructure chip was fabricated from a 10 cm silicon wafer (MEMC) with a resistivity of 10 ohm/ cm. Matrixes R-cyano-4-hydroxycinamic acid (CHCA) and 2,5dihydroxybenzoic acid (DHB) were obtained from Sigma-Aldrich (St. Louis, MO) for MALDI-TOFMS. Acetonitrile (ACN, 99.9%) was purchased from Merck (Darmstadt, Germany). Acetone, trifluoroacetic acid (TFA), and phosphoric acid (PA) were obtained from Fluka (Steinheim, Switzerland). Peptide standards monophosphopeptide (β-casein 48-63, [M + H]+ ) 2061.8043) and angiotensin I ([M + H]+ ) 1296.6853) were obtained from Sigma-Aldrich. Bovine serum albumin (BSA) digests was purchased from Waters (Milford, MA). Fabrication of the Microstructure Chip. The microstructure chip was fabricated via standard photolithography and Bosch etching (STS, Multiplex ICP) as described previously.16 The process is illustrated in Scheme 1. In brief, the silicon wafer was coated with a thin layer of AZ4620 (positive photoresist) by spin coating, followed by etching of the radiate design pattern to a depth of 15 µm. The remaining photoresist was then stripped by acetone. In order to reduce the friction of the microstructure and to minimize biomolecule adhesion, a passivation layer of plasmapolymerized fluorocarbon (PPFC) was deposited onto the chip by inductively coupled plasma (ICP) to form the hydrophobic layer. The chip size was designed according to the ABI (Applied Biosytems) MALDI sample plate holder (40 mm ×46 mm) comprising 10 × 10 wells. Each well was patterned with lines radiating from the central zone. In this study, radiate microstructure wells (5 mm in diameter) with central spaces of different diameters (500, 600, 700, 800, and 900 µm) were examined as sample anchors for MALDI sample preparation. With proper care and a clean environment, the fabricated chip can be stored for months or even years. Sample Preparation. Each peptide sample (angiotensin I and β-casein (48-63)) was dissolved in 50% acetonitrile to reach a final mixture concentration of 2 pmol/µL. For optimization and matrix homogeneity studies, different concentrations of matrixes (10, 5, 4, 2, 1.5, 1, 0.5, 0.1 mg/mL) were prepared and dissolved in 50% (15) Meng, J. C.; Siuzdak, G.; Finn, M. G. Chem. Commun. 2004, 21, 2108– 2109. (16) Lin, M. Y.; Yu, C. S.; Hu, Y. C.; Cheng, S. C.; Hu, H. T. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2005, 1, 530–533.
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ACN. All DHB and CHCA matrixes were prepared and dissolved in 50% ACN with 0.1% TFA. All peptide samples and matrixes were prepared immediately before being used. Mass Spectrometric Analysis. MALDI-TOFMS analysis was performed on a Voyager-DE Pro with a pulsed nitrogen laser at 337 nm (Applied Biosytems, Foster City, CA). For sample preparation, 1 µL of sample was mixed with an equal volume of matrix. We then deposited 1 µL of analyte solution onto a stainless steel sample plate or the microstructure chip and allowed it to air-dry at room temperature. All mass spectra were acquired in the linear positive-ion mode with delayed extraction. The extraction and guide wire voltages were set at 20 kV and 0.05%. A low mass gate was set at 1000 Da. For all MALDI-TOFMS analyses, 10 profiles containing 80 shots each from the same spots were acquired. Mass calibration was externally calibrated using ion peaks of angiotensin I, ACTH (1-17), and ACTH (7-38). The data analysis was performed by Data Explorer software (Applied Biosystems). RESULTS AND DISCUSSION Radiate Microstructure Chip. The disposable radiate microstructure chip developed in this study is depicted in Figure 1. Angiotensin I and β-casein peptides (1 µL) were mixed with equal volume of matrixes CHCA and DHB, respectively. After applying an appropriate volume of sample onto the well of the chip, we found that the droplet was confined to the center by the radiate microstructure. We had used even up to 30 µL of albumin (5 mg/ mL) as sample and finally got a dried spot confined within the central zone of the well until air-dried.17 In this study, 1 µL analyte was applied onto the steel target plate or microstructure chip separately and allowed to dry. Unlike the chemical coating methods with a hydrophilic anchor such as gold spots3 or 1-methoxy-2-propyl acetate (SILCLEAN 3700),6 the gradient wettability on the microstructure chip was generated by the radiate texture of the chip. Based on the Laplace-Young equation, the contact interface between the liquid and solid phases is associated with wettability. The central zone of the chip has the largest contact area for the droplet, which implies that it is the least hydrophobic part of the chip. Besides, the hydrophobic
Figure 1. (A) Photograph of the silicon wafer with the microstructure chip manufactured via photolithography. (B) A segmented chip was attached to the ABI sample adapter with conductive tape. (C) Droplets of water (left) and analyte (right) are seen within the central zone of the well. The upper right illustration in C shows the pattern of one microstructure spot.
Figure 2. The contact angle of water (A, B) and analyte (C) droplets were measured both on the flat (left) and microstructure (right) parts of chips with (B, C) and without (A) PPFC coating. (D) The photographs of analyte droplets were captured in time series when dried on the microstructure well of the chip. (E) The crystals that formed were observed by SEM. Both CHCA (upper) and DHB (below) were confined precisely within the central zone.
character of the PPFC coating on our chip minimizes the adhesion of unwanted peptides or analyte to the sample plate. The central zone anchors the analyte because of the different hydrophobic characteristics formed by the radiate texture. In our experiments, we found that analytes could easily be applied onto wells with central zones measuring 800 or 900 µm but not onto wells with central zones measuring less than 700 µm. The contact area, therefore, seems to play an important role in anchoring the analyte to the microstructure chip surface. Since a smaller dried zone implies a better concentration effect, the 800-µm diameter central zone was selected for further experiments involving analyte concentration and sample deposition. The contact angle of water droplets was measured on chips with and on chips without the hydrophobic PPFC layer. In chips without the PPFC coating, we found that the microstructure was more hydrophobic than the flat part of the chip (Figure 2A). Moreover, we found that the contact angle of the water droplet did not differ significantly between the flat structure and the microstructure on PPFC-coated chips (Figure 2B). However, when the analyte solution (50% ACN) was applied onto the chip, the analyte droplet as well as water was confined within the radiate microstructure because of the hydrophobic effect in our chip. In the flat parts of the chip, the droplets collapsed (Figure 2C). The results indicate that our microstructure chip provided good hydrophobic characteristics for water and organic solvent even though it lacked the PPFC coating. The analyte drying process was also performed in a time series (figure 2D). The needle-like crystals finally formed over the central space of the microstructure chip after the solvent had entirely evaporated. The crystals that formed within the 800-µm diameter central space of the well were then observed under a scanning electronic microscope (SEM)
(figure 2E). All of the pellet-like (CHCA) or needle-like (DHB) crystals were confined precisely within the central zone of the microstructure chip. Matrix Optimization of the Microstructure Chip Surface. It has been shown that preparation of a thin, homogeneous layer of sample/matrix cocrystals improves the sensitivity and mass accuracy of MALDI-TOFMS in peptides analysis.18,19 Moreover, the influence of the solvent for matrix, sample, and crystal growth time on the quality of MALDI-MS analyses has also been systematically studied.20,21 To optimize the matrix homogeneity and reproducibility in our chip, a series of concentrations (10, 5, 2, 1, 0.5, 0.1 mg/mL) of CHCA and DHB matrixes were prepared and mixed with equal volumes of angiotensin I (2 pmol/mL) and β-casein (2 pmol/mL) peptides, respectively. Then these samples were applied onto the microstructure chip for MALDI-TOFMS analysis. MALDI-MS analysis revealed an obvious difference in signal intensity between the chip and steel plate when matrix concentrations ranged from 10 to 0.1 mg/mL both in CHCA and DHB. An enhanced signal was found at a matrix concentration of 2 mg/mL for the chip (Figure 3A-D) and at 10 mg/mL for the plate (Figure 3E-H). The signal intensity of angiotensin I was 2-fold higher (Figure 3B, 3F) and that of β-casein was 4-fold higher (Figure 3D, 3H) in the microstructure chip than in the metallic sample plate. The results show that the microstructure chip economizes on the matrix during the MALDI analysis. Since the most often used concentration for traditional metallic plate is 10 mg/mL (Figure 3E, 3G), the CHCA crystal that formed within the 800 µm-diameter-chip under the same concentration (10 mg/mL) seems to be too coarse for the analyte to be efficiently ionized (Figure 3A, 3C). However, the amount of added sample was not altered, indicating that the enhanced signal intensity was due to the ability of our microstructure chip to concentrate the sample. The thickness of the crystal distribution was determined by the basal contact area. High concentration of matrix (>4 mg/ mL) decreased the sample/matrix ratio and formed a thicker crystal, thereby lowering the efficiency of laser desorption. In our experiment, we found that 2 mg/mL matrix was the optimum working concentration for our chip in MALDI/MS analysis. These results were re-examined in more than 10 independent experiments. In addition, the sample ring marked on the ABI sample plate was 2200 µm in diameter. As compared to the matrix concentration per basal area unit between the chip and metallic sample plate, we observed a similar matrix distribution ratio R (2.469 × 10-6 and 2.066 × 10-6, respectively). The ratio R was calculated using the following formula: R ) M/r2 where M is the concentration of matrix, and r is the diameter of matrix distribution. (17) Chen, S. Y.; Yu, C. S.; Wang, J. S.; Huang, C. C.; Hu, Y. C. 13th Conf. Proc. ICBME 2008, 23, 799–801. (18) Vorm, O.; Roestorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281–3287. (19) Xiang, F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199–204. (20) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31–37. (21) Jensen, C.; Haebel, S.; Andersen, S. O.; Roepstorff, P. Int. J. Mass Spectrom. Ion Processes 1997, 160, 339–356.
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Figure 3. MALDI mass peak intensity spectra obtained from various concentrations (2, 10 mg/mL) of CHCA and DHB matrix with angiotensin I ([M + H]+ ) 1296.68) and β-casein 48-63 peptides ([M + H]+ ) 2061.80), respectively. These results were obtained from the microstructure chip (A-D) and from the metallic sample plate (E-H) respectively. Panels A and E show the results from Angiotensin I mixed with 10 mg/mL CHCA, and panels B and F show the results from Angiotensin I mixed with 2 mg/mL CHCA. Panels C and G show the results of β-casein peptide mixed with 10 mg/mL DHB, and panels D and H show the results of β-casein peptide mixed with 2 mg/mL DHB.
Nonetheless, the signal intensity was still higher with the chip than with the metallic plate at a sample concentration of 10 mg/ mL. At a CHCA concentration of 0.1 mg/mL, angiotensin I could be detected with our chip (signal/noise >3) but was not detectable on the metallic plate. The results suggest that the matrix concentration and homogeneity are important factors for sensitivity of signal detection. With the optimization of the matrix concentration, our chip could effectively concentrate the sample. Since the growth rates of the sample/matrix cocrystals might influence the MALDI-MS analysis of protein and peptide,22 it was essential to find the optimum crystal-forming condition in this experiment. A series of matrix solvents (ACN:EtOH: 0.1%TFA ) 90:0:10, 80:0:20, 70:20:10, 50:20:30, 30:20:50, and 10:20:70) were used and observed under a microscope after being air-dried (data not shown). The crystal type with low ACN content had a very slow evaporation rate and formed crystals with a coarse-grained texture. Crystals with high ACN content, however, had a uniform (22) Kussmann, M.; Roepstorff, P. Methods Mol. Biol. 2000, 146, 405–424.
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distribution and a faster evaporation rate. We found that crystals that formed with high content of organic solvent were generally homogeneous on the microstructure chip and led to good sensitivity. Limit of Detection of the Microstructure Chip. The performance of the microstructure chip was studied using the optimized DHB matrix concentration (2 mg/mL) mixed with a serial dilution of monophosphopeptide β-casein stock solution (500, 100, 50, 10 fmol/µL, in 50% ACN). Figure 4A-D shows the mass spectra obtained with 500, 100, 50, and 10 fmol/µL of β-casein phosphopeptide concentrated by the microstructure chip, and Figure 4E-H shows the mass spectra obtained by the metallic plate. When the low mass gate was set at 1000 Da, the signal/noise ratio of 5 fmol/µL β-casein phosphopeptide was 98.2. Based on a S/N ratio of 3, we estimated that the limit of detection (LOD) for phosphopeptide on our microstructure chip was 150 attomoles. In addition, the same experimental conditions were also applied to the metallic sample plate for comparative purposes. The signal intensity of β-casein phos-
Figure 4. Mass spectra of β-casein phosphopeptide analyzed on the microstructure chip (A-D) and metallic plate (E-H). (A-D) The mass spectra obtained with 500 (A, E), 100 (B, F), 50 (C, G), and 10 (D, H) fmol/µL of β-casein peptide, respectively. Panels A-D show the results from β-casein phosphopeptide mixed with 2 mg/mL DHB. Panels E-H show the results of β-casein peptide mixed with 10 mg/mL DHB.
phopeptide was not detectable at 10 fmol/µL (low mass gate on). For each concentration of β-casein phosphopeptide, the signal intensities obtained from the microstructure chip were greater than those obtained from the metallic control. From these results, it is clear that concentration of the analyte on the microstructure chip improves the sensitivity of detection. Analysis of BSA Digests on Microstructure Chip. To examine whether the microstructure chip is suitable for multiple peptide analysis, 1 pmol/µL of BSA digests (in 50% ACN) was mixed with DHB matrix (1:1 v/v) and then analyzed on the microstructure chip and on the metallic plate by MALDI-TOFMS. Compared with the metallic plate, the signal intensities from the microstructure chip were higher as shown in Figure 5. Moreover, the relative intensity pattern between various peptides differed between the chip and the plate. Several peak intensities were significantly elevated in the microstructure chip (m/z 506.34, 688.71, 1418.72, 1479.94, 1880.36, 2493.24). The results suggest that these elevated peak intensities resulted from the ability of our microstructure chip to concentrate the sample. Therefore, our chip may improve the detection rate of some rare peptides in protein digests. Analysis of Phosphopeptide in BSA Digests. To assess the feasibility of using this microstructure chip in the laboratory, 1 pmol/µL of BSA digests (50% acetonitrile) was mixed with
Figure 5. Mass spectra of BSA digests (1 pmol/mL, 50% acetonitrile) analyzed on microstructure chip (A) and metallic plate (B). The asterisks indicate the peaks that are significantly elevated on the chip.
equal volumes of β-casein monophosphopeptide in a series of concentrations (5, 2, 1, 0.5, 0.2, 0.1 pmol/µL). In general, phosphoproteins are characterized by MALDI-TOFMS after enzyme digestion, but the signals of the phosphopeptides are Analytical Chemistry, Vol. 82, No. 14, July 15, 2010
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Figure 6. MALDI-TOFMS spectra obtained from the various concentrations of β-casein monophosphopeptide mixed with BSA digests. (A, E) 1 pmol/µL, (B, F) 500 fmol/µL, (C, G) 200 fmol/µL, and (D, H) 100 fmol/µL of β-casein monophosphopeptide were mixed with equal volumes of BSA digests. These analytes were analyzed with (A-D) the microstructure chip, or (E-H) with a metallic sample plate. The black arrow shows the β-casein phosphopeptide 48-63, [M + H]+ ) 2061.8043.
always suppressed by the presence of other peptides. We used mimic mixtures to examine the performance of the microstructure chip to detect phosphopeptide in mixtures. Similar mass spectra were found both in the metallic plate and the chip at concentrations ranging from 5 pmol/µL to 1 pmol/µL. However, the sample concentrated on the chip yielded higher signal intensities than that on the metallic plate at each concentration (Figure 6). However, in MALDI-TOFMS analysis, the matrix signal is usually much higher than the sample signal. Therefore, to increase the signal-to-noise ratio for both plate and chip assays, a low mass gate was set at 1000 Da. In this restricted 5956
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Table 1. Signal-to-Noise Ratios in Phosphopeptide Detection for Microstructure Chip and Metallic Plate phosphopeptide (pmol/µL) S/N ratio:chip S/N ratio:plate
5
2
1
0.5
0.2
0.1
857.4 248.2
504.1 105.1
205.7 61.9
130.1 55.1
48.9 N/A
N/A N/A
mass gate, even at a concentration of 200 fmol/µL, the chip revealed significantly higher signal-to-noise ratios in phosphopeptide detection (see Table 1).
CONCLUSIONS Our silicon-based microstructure chip enhances the signal intensity of MALDI-MS by concentrating the analyte. Appropriate solvent proportion and matrix concentration yielded a homogeneous distribution of crystal, thereby improving the sensitivity of MALDI-MS. In addition, β-casein monophosphopeptide mixed with BSA-digest sample was also examined to assess the suppression effect of non-phosphopeptides. With the properties of easy fabrication, disposability, and on-target concentration of MALDI-MS samples, the microstructure chip
represents an alternative method for high-throughput proteomics research. ACKNOWLEDGMENT The study was funded by a grant from the National Research Council of the Republic of China. Received for review January 28, 2010. Accepted June 7, 2010. AC101426N
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