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Cite this: Chem. Commun., 2014, 50, 4831 Received 20th February 2014, Accepted 17th March 2014
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Detection and quantification of the Bcr/Abl chimeric protein on biochips using LDI-TOF MS† Seol-Hye Hong,a Jae Il Kim,a Hyunook Kang,a Soojin Yoon,b Dong-Eun Kim,b Woong Jungc and Woon-Seok Yeo*a
DOI: 10.1039/c4cc01332h www.rsc.org/chemcomm
The Bcr/Abl chimeric protein was captured by two antibodies, anti-Bcr on gold nanoparticles (AuNPs) and anti-Abl on a biochip, in a sandwich assay format. The presence of the Bcr/Abl in cells was then verified by amplified LDI-TOF MS signals, and relative amounts were quantified using AuNPs coated with deuterated alkanethiols as an internal standard.
Chronic myelogenous leukemia (CML) is a hematological disorder characterized by the presence of the Philadelphia chromosome, which is derived from a translocation between chromosome 9 and chromosome 22 that results in a Bcr/Abl fusion gene.1 The expressed Bcr/Abl chimeric protein from the Philadelphia chromosome induces abnormal signaling, leading to suppression of apoptosis and increased proliferation and survival of cells, and is required for the pathogenesis of CML.2 Thus, the Bcr/Abl chimeric protein is both an attractive target for therapeutic invention and a biomarker for the diagnosis of CML.3 In addition, the Bcr/Abl chimeric protein expression level is associated with CML progression and the response to therapeutic treatment, and therefore, accurate quantification of the Bcr/Abl protein would be significant for assessing prognosis after therapeutic treatment.4 For these reasons, various detection and quantitation methods for Bcr/Abl have been reported, including fluorescence from in situ hybridization,5 the real-time polymerase chain reaction,6 electrochemical-based biosensors,7 and methods based on gold nanoparticles (AuNPs) or quantum dots.8 However, these methods targeted the genetic or transcriptional level of Bcr/Abl, and not the Bcr/Abl chimeric protein, which is the real disease-inducing factor. In many cases, gene expression is interpreted in terms of protein levels; however, the correlation is not strong and can be as little as 40% depending on the system, a
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea. E-mail: [email protected] b Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea c Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, Korea † Electronic supplementary information (ESI) available: Experimental details and additional data described in the text. See DOI: 10.1039/c4cc01332h
This journal is © The Royal Society of Chemistry 2014
implying that the mRNA quantity may not represent the real amount of the corresponding protein.9 Therefore, more effective detection and quantification of the expressed Bcr/Abl chimeric protein can provide higher diagnostic and prognostic accuracy. Traditionally and most commonly, western blot analysis (also called immunoblotting) is the most powerful protein quantification method. Although many researchers have been using western blotting which can provide sensitive and selective detection and quantitative information, its general use is sometimes hampered by a high level of background signals when detecting proteins at very low concentrations, a time-consuming and complicated experimental process, and necessity of a trained expert.10 Here, we introduce a protein detection and quantification method employing a rapid and simple experimental protocol that retains the selectivity and sensitivity of western blotting, using self-assembled monolayers on gold and laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS) with a matrix-free format. The strategy for detection and quantification of Bcr/Abl chimeric proteins on biochips relies on mass signal amplification using AuNPs coated with small molecules called Am-tags (amplification tags).11 As shown in Scheme 1, the Bcr/Abl chimeric protein in the cell lysate is captured by two antibodies, anti-Bcr on AuNPs and antiAbl on biochips in a sandwich assay format. The captured target chimeric protein is subsequently verified by LDI-TOF MS signals of the Am-tag on AuNPs. The Am-tag, a small molecule excessively decorated on AuNPs, plays two critical roles: (1) mass signal amplification, and (2) prevention of non-specific absorption on AuNPs.12 To quantify the captured proteins, we introduced AuNPs decorated with the deuterium-substituted Am-tag (dAm-tag) as an internal standard (IS). The dAm-tag has the same molecular structure as the Am-tag except for the replacement of four hydrogens with deuteriums at two carbons.13 The dAm-tag would be identical to the Am-tag with respect to desorption/ionization and other handling procedures except for the molecular weight. Therefore, comparison of mass intensities of the Am-tag with that of the dAm-tag allows estimation of the relative amount of the captured Bcr/Abl chimeric protein on the biochip. The structures of Am-tag-coated AuNPs, dAm-tag-coated AuNPs, and the biochip used in this study are
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Scheme 1 Schematic diagram of detection and quantification of the Bcr/Abl chimeric protein using MALDI-TOF MS.
Fig. 1 The structures and MS verification of (a) Am-tag-coated AuNP, (b) dAm-tag-coated AuNP, and (c) the biochip used in this study.
shown in Fig. 1. Tri(ethylene glycol) groups on both AuNPs and chips provide inertness, i.e. prevent non-specific protein adsorption on the surface. Particularly, our strategy avoids the use of an organic matrix, which in general interferes with analysis in the low mass region, and therefore, can afford clear signals of Am-tag and dAm-tag without any significant background noise.14 For a model cell line, we used the Ba/F3 cell line which is a mouse B lymphocyte cell line and is well known to express Bcr/Abl chimeric proteins. First, we prepared Am-tag-coated AuNPs and dAm-tag-coated AuNPs, and analyzed using LDI-TOF MS. AuNPs (40 nm in diameter, 1 mL of 3.3 nM) were incubated with a mixed solution (100 mM in ethanol) of tri(ethylene glycol)-terminated thiol and carboxylic penta(ethylene glycol)-terminated thiol in a ratio of 95 : 5 for 12 h. LDI-TOF MS analysis of the resulting AuNPs showed peaks at m/z 553.3 [M + Na]+ and 569.3 [M + K]+, corresponding to tri(ethylene glycol)-containing disulfides, and at m/z 699.4 [M + Na]+, corresponding to carboxylic acid-containing disulfide, verifying the Am-tag and the acid functionality on AuNPs (Fig. 1a, bottom). For the preparation of the IS (i.e. dAm-tag-coated AuNPs), AuNPs were incubated with a solution (100 mM in ethanol) of deuteriumsubstituted tri(ethylene glycol)-terminated thiols for 12 h. The mass analysis of these AuNPs showed a major peak at m/z 561.3 [M + Na]+, corresponding to deuterium-substituted tri(ethylene glycol)-containing disulfide (Fig. 1b, bottom). Using the same experimental procedure, carboxyl group-presenting biochips were prepared in a mixed solution (1 mM in ethanol) of a tri(ethylene glycol)-terminated alkanethiol and
4832 | Chem. Commun., 2014, 50, 4831--4834
ChemComm
a carboxylic penta(ethylene glycol)-terminated alkanethiol in a ratio of 95 : 5 for 12 h. Note that alkanethiols with eleven-carbon alkyl chains were used for the preparation of chips. The mass spectrum of the chip showed peaks at m/z 693.4 [M + Na]+ and 709.4 [M + K]+, corresponding to the tri(ethylene glycol) containing disulfides, and at m/z 839.6 [M + Na]+, corresponding to the carboxylic acid-containing disulfide (Fig. 1c, bottom). Next, we examined the selective capture and detection of the Bcr/Abl chimeric protein in a complex sample using our strategy. For the immobilization of antibodies on AuNPs and gold chips to capture the Bcr/Abl chimeric protein, carboxylic acid groups on the surfaces were activated with NHS and EDC and the resulting NHS-activated surfaces were then incubated with antibodies (0.87 mM anti-Abl and 1.03 mM anti-Bcr in PB) for 1 h. Ba/F3 cells were lysed by sonication and the cell lysate was incubated with antiBcr-immobilized AuNPs. The AuNPs were separated by centrifugation, resuspended in PB, and treated on anti-Abl immobilized gold chips. The chips were rinsed with distilled water to remove unbound AuNPs and analyzed by LDI-TOF MS. As shown in Fig. S1a (ESI†), a peak at m/z 553 corresponding to the Am-tag was clearly observed with a high signal-to-noise ratio, indicating that Bcr/Abl proteins in the lysed cell mixture were captured by the two antibodies on the AuNP and the chip. As controls, anti-PSA was used instead of antiBcr on AuNPs and instead of anti-Abl on the chip, and these systems did not produce the peak at m/z 553 (Fig. S1b and 1c, ESI†). Taken together, these results imply that Bcr/Abl proteins in a complex sample were captured by our strategy with high selectivity, as described in Scheme 1, without any significant background noise. Next, we assessed the feasibility of relative quantification of Bcr/Abl proteins in cells using the IS. Lysed cell supernatants from various numbers of cells were incubated with anti-Bcr-presenting AuNPs for 30 min. The AuNPs were separated by centrifugation, resuspended, and treated on the anti-Abl-presenting biochip for 30 min. The chip was rinsed with distilled water to remove unbound AuNPs, treated with the IS, and dried under ambient conditions. The chip was then transferred to a PCR tube containing 200 mL of ethanol and subjected to sonication for 30 s to detach the AuNPs and IS from the chip. In this way, surface-bound AuNPs and the IS were homogenized to avoid spot-to-spot variation during LDI-TOF MS analysis. The AuNPs and the IS were separated by centrifugation, resuspended in ethanol, and analyzed by LDI-TOF MS without a matrix. Fig. 2a shows the typical mass spectra of the detection of Bcr/Abl in lysed cells in the presence of the IS in various numbers of lysed cells ranging from 0.3 104 to 1.6 104. As expected, the intensities of the Am-tag gradually increased as the number of lysed cells increased. Note that the spectra in Fig. 2a were normalized by the sum of Am-tag and dAm-tag peaks to enable clear visualization of the gradual increase in Am-tag signals as the number of lysed cells increased.15 In the control experiment using anti-PSA-immobilized AuNPs, only dAm-tag signals were observed (Fig. 2b). As another control, we used naı¨ve Ba/F3 cells which do not express the Bcr/Abl protein and, as expected, only a trace of Am-tag signals were observed (Fig. 2c). These two control experiments clearly indicate not only selective capture of target proteins but also essentially no false-positive signals using our strategy regardless of the number of lysed cells.
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Fig. 2 (a) Typical mass spectra of the detection of Bcr/Abl in lysed cells in the presence of the IS in various numbers of lysed cells ranging from 0.3 104 to 1.6 104. (b) Control experiment using anti-PSA-coated AuNPs. (c) Control experiment using naı¨ve Ba/F3 cell lysates.
We constructed the calibration curve by a linear regression of the ratio of signal intensities between Am-tag and dAm-tag in Fig. 2a against the number of lysed cells. The calibration curve shows that linearity occurred over the number of lysed cells ranging from 0.3 104 to 1.6 104 (Fig. 3a). It is widely accepted that MALDI-TOF MS is not inherently quantitative and direct use of mass intensity for quantification is not practical because of poor shot-to-shot and sample-to-sample reproducibility.16 As such, this linearity clearly verifies the fidelity of our strategy for quantification of target proteins using the dAm-tag-coated AuNPs as an IS, as shown in Scheme 1. For further verification of reliability of our strategy, we performed a western blot analysis which is the most common method for relative protein quantification. Fig. 3b shows the western blot analysis of the Bcr/Abl chimeric protein in Ba/F3 cells using anti-Abl and b-actin as a control. A major band at 210 kDa corresponds to the Bcr/Abl chimeric protein and this becomes gradually thicker as the number of lysed cells increases. Note that a band at B120 kDa stems from the c-Abl protein which is encoded by chromosome 22 in most mammalian cells.17 In the western blotting, b-actin may not play a role of an internal standard for comparing the relative quantity of a protein of interest in this system because of the use of two different probes, anti-Abl and anti-b-actin, and the non-specific adsorption of these antibodies when using cell lysates from a larger number of cells.
Fig. 3 (a) The calibration curve constructed by a linear regression of the ratio of signal intensities between Am-tag and dAm-tag in Fig. 2a against the number of lysed cells. The standard deviations were obtained from three independent experiments. (b) Western blot analysis of the Bcr/Abl chimeric protein expression in Ba/F3 cells and the ratios of band intensities between the Bcr/Abl protein and b-actin at various cell numbers. b-actin was used as a control.
This journal is © The Royal Society of Chemistry 2014
Communication
Compared with the western blotting, our strategy is beneficial for detecting and quantifying Bcr/Abl chimeric proteins. Firstly, the experimental process of western blotting is complicated and time-consuming, because it requires many consecutive steps including electrophoresis, membrane transfer, staining, and blocking. In addition, deleterious non-specific protein adsorption often causes a high level of background signals and false-positive signals which may interfere with the accurate interpretation of the data when using western blotting to analyze proteins with low abundance. By contrast, our approach takes only a couple of hours, except for formation of the self-assembled monolayer and antibody immobilization on the AuNPs and gold chips. These steps do not require a high degree of skill. Furthermore, our approach affords substantially low background signals and falsepositive signals, resulting in enhanced selectivity. This is attributed to oligo(ethylene glycol) groups on the AuNPs and the chips (see Fig. 1 and 2). Finally, sensitivity of our strategy could be further improved by grafting miniaturization systems such as microfluidics or a lab-on-a-chip device. In summary, we have demonstrated a rapid and simple strategy for detecting and quantifying the Bcr/Abl chimeric protein in a complex sample using LDI-TOF MS. This protein was selectively captured by two immobilized antibodies on AuNPs and a biochip in a sandwich assay format. The presence of the protein was verified by the amplified mass signal of small molecules called Am-tags on the AuNPs. Our strategy clearly detected the Bcr/Abl chimeric protein in cell lysates, as evidenced by Am-tag signals of LDI-TOF MS. Furthermore, we successfully quantified the relative amount of the Bcr/Abl chimeric protein in the lysed cell mixture by using deuterated Am-tag-coated AuNPs as an internal standard. We believe that our strategy will be a useful tool for diagnosis and prognosis after treatment of chronic myeloid leukemia. This research was supported by the Basic Science Research Program (NRF-2013R1A1A2007272) and the Priority Research Centers Program (2009-0093824) through the National Research Foundation (NRF) of Korea funded by the Ministry of Education.
Notes and references 1 A. de Klein, A. G. van Kessel, G. Grosveld, C. R. Bartram, A. Hagemeijer, D. Bootsma, N. K. Spurr, N. Heisterkamp, J. Groffen and J. R. Stephenson, Nature, 1982, 300, 765; G. Q. Daley, R. A. Van Etten and D. Baltimore, Science, 1990, 247, 824; F. Mitelman, B. Johansson and F. Mertens, Nat. Rev. Cancer, 2007, 7, 233. 2 L. Puil, J. Liu, G. Gish, G. Mbamalu, D. Bowtell, P. G. Pelicci, R. Arlinghaus and T. Pawson, EMBO J., 1994, 13, 764; D. Cortez, G. Reuther and A. M. Pendergast, Oncogene, 1997, 15, 2333; N. N. Danial and P. Rothman, Oncogene, 2000, 19, 2523; R. Ren, Nat. Rev. Cancer, 2005, 5, 172. 3 A. S. Advani and A. M. Pendergast, Leuk. Res., 2002, 26, 713. 4 H. Modi, T. McDonald, S. Chu, J. K. Yee, S. J. Forman and R. Bhatia, Blood, 2007, 109, 5411. 5 G. W. Dewald, W. A. Wyatt, A. L. Juneau, R. O. Carlson, A. R. Zinsmeister, S. M. Jalal, J. L. Spurbeck and R. T. Silver, Blood, 1998, 91, 3357. 6 T. Burmeister and R. Reinhardt, Leuk. Res., 2008, 32, 579. 7 K. Wang, J. Chen, J. Chen, A. Liu, G. Li, H. Luo, X. Lin and Y. Chen, Electroanalysis, 2009, 21, 1159; L. Lin, J. Kang, S. Weng, J. Chen, A. Liu, X. Lin and Y. Chen, Sens. Actuators, B, 2011, 155, 1. 8 K. Jangpatarapongsa, D. Polpanich, V. Yamkamon, Y. Dittharot, J. Peng-On, R. Thiramanas, S. Hongeng, S. Jootar, L. Charoenmak
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9 10 11 12 13
and P. Tangboriboonrat, Analyst, 2011, 136, 354; A. Sharma, G. Sumana, S. Sapra and B. D. Malhotra, Langmuir, 2013, 29, 8753. C. Vogel and E. M. Marcotte, Nat. Rev. Genet., 2012, 13, 227. B. T. Kurien and R. H. Scofield, Methods, 2006, 38, 283. J. R. Lee, J. Lee, S. K. Kim, K. P. Kim, H. S. Park and W.-S. Yeo, Angew. Chem., Int. Ed., 2008, 47, 9518; H. Seo, S. Kim, J. I. Kim, H. Kang, W. Jung and W.-S. Yeo, Anal. Biochem., 2012, 434, 199–201. M. Mrksich and G. M. Whitesides, ACS Symp. Ser., 1997, 680, 361. The synthetic scheme and experimental details for the dAm-tag will be published elsewhere.
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ChemComm 14 J. Lee, J. Lee, T. D. Chung and W.-S. Yeo, Anal. Chim. Acta, 2012, 736, 1. 15 During the MS acquisition, a gold cluster peak (Au3) was found at m/z 591. The intensity of that peak was not significantly high, and therefore, did not induce the signal suppression or any other effect on the Am-tag signals. 16 J. Albrethsen, Clin. Chem., 2007, 53, 852; E. Szajli, T. Feher and K. F. Medzihradszky, Mol. Cell. Proteomics, 2008, 7, 2410. 17 A. Sirvent, C. Benistant and S. Roche, Biol. Cell, 2008, 100, 617.
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Cite this: Chem. Commun., 2014, 50, 4831 Received 20th February 2014, Accepted 17th March 2014
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Detection and quantification of the Bcr/Abl chimeric protein on biochips using LDI-TOF MS† Seol-Hye Hong,a Jae Il Kim,a Hyunook Kang,a Soojin Yoon,b Dong-Eun Kim,b Woong Jungc and Woon-Seok Yeo*a
DOI: 10.1039/c4cc01332h www.rsc.org/chemcomm
The Bcr/Abl chimeric protein was captured by two antibodies, anti-Bcr on gold nanoparticles (AuNPs) and anti-Abl on a biochip, in a sandwich assay format. The presence of the Bcr/Abl in cells was then verified by amplified LDI-TOF MS signals, and relative amounts were quantified using AuNPs coated with deuterated alkanethiols as an internal standard.
Chronic myelogenous leukemia (CML) is a hematological disorder characterized by the presence of the Philadelphia chromosome, which is derived from a translocation between chromosome 9 and chromosome 22 that results in a Bcr/Abl fusion gene.1 The expressed Bcr/Abl chimeric protein from the Philadelphia chromosome induces abnormal signaling, leading to suppression of apoptosis and increased proliferation and survival of cells, and is required for the pathogenesis of CML.2 Thus, the Bcr/Abl chimeric protein is both an attractive target for therapeutic invention and a biomarker for the diagnosis of CML.3 In addition, the Bcr/Abl chimeric protein expression level is associated with CML progression and the response to therapeutic treatment, and therefore, accurate quantification of the Bcr/Abl protein would be significant for assessing prognosis after therapeutic treatment.4 For these reasons, various detection and quantitation methods for Bcr/Abl have been reported, including fluorescence from in situ hybridization,5 the real-time polymerase chain reaction,6 electrochemical-based biosensors,7 and methods based on gold nanoparticles (AuNPs) or quantum dots.8 However, these methods targeted the genetic or transcriptional level of Bcr/Abl, and not the Bcr/Abl chimeric protein, which is the real disease-inducing factor. In many cases, gene expression is interpreted in terms of protein levels; however, the correlation is not strong and can be as little as 40% depending on the system, a
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea. E-mail: [email protected] b Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea c Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, Korea † Electronic supplementary information (ESI) available: Experimental details and additional data described in the text. See DOI: 10.1039/c4cc01332h
This journal is © The Royal Society of Chemistry 2014
implying that the mRNA quantity may not represent the real amount of the corresponding protein.9 Therefore, more effective detection and quantification of the expressed Bcr/Abl chimeric protein can provide higher diagnostic and prognostic accuracy. Traditionally and most commonly, western blot analysis (also called immunoblotting) is the most powerful protein quantification method. Although many researchers have been using western blotting which can provide sensitive and selective detection and quantitative information, its general use is sometimes hampered by a high level of background signals when detecting proteins at very low concentrations, a time-consuming and complicated experimental process, and necessity of a trained expert.10 Here, we introduce a protein detection and quantification method employing a rapid and simple experimental protocol that retains the selectivity and sensitivity of western blotting, using self-assembled monolayers on gold and laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS) with a matrix-free format. The strategy for detection and quantification of Bcr/Abl chimeric proteins on biochips relies on mass signal amplification using AuNPs coated with small molecules called Am-tags (amplification tags).11 As shown in Scheme 1, the Bcr/Abl chimeric protein in the cell lysate is captured by two antibodies, anti-Bcr on AuNPs and antiAbl on biochips in a sandwich assay format. The captured target chimeric protein is subsequently verified by LDI-TOF MS signals of the Am-tag on AuNPs. The Am-tag, a small molecule excessively decorated on AuNPs, plays two critical roles: (1) mass signal amplification, and (2) prevention of non-specific absorption on AuNPs.12 To quantify the captured proteins, we introduced AuNPs decorated with the deuterium-substituted Am-tag (dAm-tag) as an internal standard (IS). The dAm-tag has the same molecular structure as the Am-tag except for the replacement of four hydrogens with deuteriums at two carbons.13 The dAm-tag would be identical to the Am-tag with respect to desorption/ionization and other handling procedures except for the molecular weight. Therefore, comparison of mass intensities of the Am-tag with that of the dAm-tag allows estimation of the relative amount of the captured Bcr/Abl chimeric protein on the biochip. The structures of Am-tag-coated AuNPs, dAm-tag-coated AuNPs, and the biochip used in this study are
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Scheme 1 Schematic diagram of detection and quantification of the Bcr/Abl chimeric protein using MALDI-TOF MS.
Fig. 1 The structures and MS verification of (a) Am-tag-coated AuNP, (b) dAm-tag-coated AuNP, and (c) the biochip used in this study.
shown in Fig. 1. Tri(ethylene glycol) groups on both AuNPs and chips provide inertness, i.e. prevent non-specific protein adsorption on the surface. Particularly, our strategy avoids the use of an organic matrix, which in general interferes with analysis in the low mass region, and therefore, can afford clear signals of Am-tag and dAm-tag without any significant background noise.14 For a model cell line, we used the Ba/F3 cell line which is a mouse B lymphocyte cell line and is well known to express Bcr/Abl chimeric proteins. First, we prepared Am-tag-coated AuNPs and dAm-tag-coated AuNPs, and analyzed using LDI-TOF MS. AuNPs (40 nm in diameter, 1 mL of 3.3 nM) were incubated with a mixed solution (100 mM in ethanol) of tri(ethylene glycol)-terminated thiol and carboxylic penta(ethylene glycol)-terminated thiol in a ratio of 95 : 5 for 12 h. LDI-TOF MS analysis of the resulting AuNPs showed peaks at m/z 553.3 [M + Na]+ and 569.3 [M + K]+, corresponding to tri(ethylene glycol)-containing disulfides, and at m/z 699.4 [M + Na]+, corresponding to carboxylic acid-containing disulfide, verifying the Am-tag and the acid functionality on AuNPs (Fig. 1a, bottom). For the preparation of the IS (i.e. dAm-tag-coated AuNPs), AuNPs were incubated with a solution (100 mM in ethanol) of deuteriumsubstituted tri(ethylene glycol)-terminated thiols for 12 h. The mass analysis of these AuNPs showed a major peak at m/z 561.3 [M + Na]+, corresponding to deuterium-substituted tri(ethylene glycol)-containing disulfide (Fig. 1b, bottom). Using the same experimental procedure, carboxyl group-presenting biochips were prepared in a mixed solution (1 mM in ethanol) of a tri(ethylene glycol)-terminated alkanethiol and
4832 | Chem. Commun., 2014, 50, 4831--4834
ChemComm
a carboxylic penta(ethylene glycol)-terminated alkanethiol in a ratio of 95 : 5 for 12 h. Note that alkanethiols with eleven-carbon alkyl chains were used for the preparation of chips. The mass spectrum of the chip showed peaks at m/z 693.4 [M + Na]+ and 709.4 [M + K]+, corresponding to the tri(ethylene glycol) containing disulfides, and at m/z 839.6 [M + Na]+, corresponding to the carboxylic acid-containing disulfide (Fig. 1c, bottom). Next, we examined the selective capture and detection of the Bcr/Abl chimeric protein in a complex sample using our strategy. For the immobilization of antibodies on AuNPs and gold chips to capture the Bcr/Abl chimeric protein, carboxylic acid groups on the surfaces were activated with NHS and EDC and the resulting NHS-activated surfaces were then incubated with antibodies (0.87 mM anti-Abl and 1.03 mM anti-Bcr in PB) for 1 h. Ba/F3 cells were lysed by sonication and the cell lysate was incubated with antiBcr-immobilized AuNPs. The AuNPs were separated by centrifugation, resuspended in PB, and treated on anti-Abl immobilized gold chips. The chips were rinsed with distilled water to remove unbound AuNPs and analyzed by LDI-TOF MS. As shown in Fig. S1a (ESI†), a peak at m/z 553 corresponding to the Am-tag was clearly observed with a high signal-to-noise ratio, indicating that Bcr/Abl proteins in the lysed cell mixture were captured by the two antibodies on the AuNP and the chip. As controls, anti-PSA was used instead of antiBcr on AuNPs and instead of anti-Abl on the chip, and these systems did not produce the peak at m/z 553 (Fig. S1b and 1c, ESI†). Taken together, these results imply that Bcr/Abl proteins in a complex sample were captured by our strategy with high selectivity, as described in Scheme 1, without any significant background noise. Next, we assessed the feasibility of relative quantification of Bcr/Abl proteins in cells using the IS. Lysed cell supernatants from various numbers of cells were incubated with anti-Bcr-presenting AuNPs for 30 min. The AuNPs were separated by centrifugation, resuspended, and treated on the anti-Abl-presenting biochip for 30 min. The chip was rinsed with distilled water to remove unbound AuNPs, treated with the IS, and dried under ambient conditions. The chip was then transferred to a PCR tube containing 200 mL of ethanol and subjected to sonication for 30 s to detach the AuNPs and IS from the chip. In this way, surface-bound AuNPs and the IS were homogenized to avoid spot-to-spot variation during LDI-TOF MS analysis. The AuNPs and the IS were separated by centrifugation, resuspended in ethanol, and analyzed by LDI-TOF MS without a matrix. Fig. 2a shows the typical mass spectra of the detection of Bcr/Abl in lysed cells in the presence of the IS in various numbers of lysed cells ranging from 0.3 104 to 1.6 104. As expected, the intensities of the Am-tag gradually increased as the number of lysed cells increased. Note that the spectra in Fig. 2a were normalized by the sum of Am-tag and dAm-tag peaks to enable clear visualization of the gradual increase in Am-tag signals as the number of lysed cells increased.15 In the control experiment using anti-PSA-immobilized AuNPs, only dAm-tag signals were observed (Fig. 2b). As another control, we used naı¨ve Ba/F3 cells which do not express the Bcr/Abl protein and, as expected, only a trace of Am-tag signals were observed (Fig. 2c). These two control experiments clearly indicate not only selective capture of target proteins but also essentially no false-positive signals using our strategy regardless of the number of lysed cells.
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Fig. 2 (a) Typical mass spectra of the detection of Bcr/Abl in lysed cells in the presence of the IS in various numbers of lysed cells ranging from 0.3 104 to 1.6 104. (b) Control experiment using anti-PSA-coated AuNPs. (c) Control experiment using naı¨ve Ba/F3 cell lysates.
We constructed the calibration curve by a linear regression of the ratio of signal intensities between Am-tag and dAm-tag in Fig. 2a against the number of lysed cells. The calibration curve shows that linearity occurred over the number of lysed cells ranging from 0.3 104 to 1.6 104 (Fig. 3a). It is widely accepted that MALDI-TOF MS is not inherently quantitative and direct use of mass intensity for quantification is not practical because of poor shot-to-shot and sample-to-sample reproducibility.16 As such, this linearity clearly verifies the fidelity of our strategy for quantification of target proteins using the dAm-tag-coated AuNPs as an IS, as shown in Scheme 1. For further verification of reliability of our strategy, we performed a western blot analysis which is the most common method for relative protein quantification. Fig. 3b shows the western blot analysis of the Bcr/Abl chimeric protein in Ba/F3 cells using anti-Abl and b-actin as a control. A major band at 210 kDa corresponds to the Bcr/Abl chimeric protein and this becomes gradually thicker as the number of lysed cells increases. Note that a band at B120 kDa stems from the c-Abl protein which is encoded by chromosome 22 in most mammalian cells.17 In the western blotting, b-actin may not play a role of an internal standard for comparing the relative quantity of a protein of interest in this system because of the use of two different probes, anti-Abl and anti-b-actin, and the non-specific adsorption of these antibodies when using cell lysates from a larger number of cells.
Fig. 3 (a) The calibration curve constructed by a linear regression of the ratio of signal intensities between Am-tag and dAm-tag in Fig. 2a against the number of lysed cells. The standard deviations were obtained from three independent experiments. (b) Western blot analysis of the Bcr/Abl chimeric protein expression in Ba/F3 cells and the ratios of band intensities between the Bcr/Abl protein and b-actin at various cell numbers. b-actin was used as a control.
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Communication
Compared with the western blotting, our strategy is beneficial for detecting and quantifying Bcr/Abl chimeric proteins. Firstly, the experimental process of western blotting is complicated and time-consuming, because it requires many consecutive steps including electrophoresis, membrane transfer, staining, and blocking. In addition, deleterious non-specific protein adsorption often causes a high level of background signals and false-positive signals which may interfere with the accurate interpretation of the data when using western blotting to analyze proteins with low abundance. By contrast, our approach takes only a couple of hours, except for formation of the self-assembled monolayer and antibody immobilization on the AuNPs and gold chips. These steps do not require a high degree of skill. Furthermore, our approach affords substantially low background signals and falsepositive signals, resulting in enhanced selectivity. This is attributed to oligo(ethylene glycol) groups on the AuNPs and the chips (see Fig. 1 and 2). Finally, sensitivity of our strategy could be further improved by grafting miniaturization systems such as microfluidics or a lab-on-a-chip device. In summary, we have demonstrated a rapid and simple strategy for detecting and quantifying the Bcr/Abl chimeric protein in a complex sample using LDI-TOF MS. This protein was selectively captured by two immobilized antibodies on AuNPs and a biochip in a sandwich assay format. The presence of the protein was verified by the amplified mass signal of small molecules called Am-tags on the AuNPs. Our strategy clearly detected the Bcr/Abl chimeric protein in cell lysates, as evidenced by Am-tag signals of LDI-TOF MS. Furthermore, we successfully quantified the relative amount of the Bcr/Abl chimeric protein in the lysed cell mixture by using deuterated Am-tag-coated AuNPs as an internal standard. We believe that our strategy will be a useful tool for diagnosis and prognosis after treatment of chronic myeloid leukemia. This research was supported by the Basic Science Research Program (NRF-2013R1A1A2007272) and the Priority Research Centers Program (2009-0093824) through the National Research Foundation (NRF) of Korea funded by the Ministry of Education.
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