NANO LETTERS
Novel Application of Carbon Nanotubes for Improving Resolution in Detecting Human Serum Proteins with Native Polyacrylamide Gel Electrophoresis
2009 Vol. 9, No. 4 1320-1324
Yandong Guo,† Lingyun Huang,‡ Willy R. G. Baeyens,§ Joris R. Delanghe,| Dacheng He,‡ and Jin Ouyang*,† College of Chemistry, Beijing Normal UniVersity, Institute of Cell Biology, Beijing Normal UniVersity, Beijing 100875, People’s Republic of China, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent UniVersity, Harelbekestraat 72, B-9000 Ghent, Belgium, and Department of Clinical Chemistry, Microbiology and Immunology, UniVersity Hospital, Ghent, Belgium Received September 26, 2008; Revised Manuscript Received January 20, 2009
ABSTRACT This paper describes a novel application of carbon nanotubes for improving the resolution of a native PAGE in the detection of human serum proteins. Carbon nanotubes were functionalized and introduced into the gel of native PAGE system, and the electropherogram showed sharp, clear bands. Furthermore, the separation of some most important proteins was improved, and the established method could be applied for the detection of sera from patients with liver diseases.
Human serum is a very complex mixture of proteins that are useful diagnostic tools; alteration of the expression of some serum proteins is an early sign of an altered physiology and may be indicative of disease.1 Initial determination of the native physicochemical properties and biological functions of individual proteins from serum is frequently accomplished by the use of native polyacrylamide gel electrophoresis (PAGE),2 which continues to be one of the most powerful separation techniques for complicated biological samples.3 However, using this method, it may be difficult to isolate those proteins that possess similar sizes and charge/ mass ratios. In addition, intrinsic limitations of native PAGE, such as the interference of the high-abundance proteins like immunoglobulin G (IgG), do not allow sufficient analysis capacity for some low-abundance proteins in such complex protein mixtures as the serum proteome. Therefore, native PAGE analysis is developing toward resolution improvement and reduction of high-abundance proteins interferences. Since their discovery in 1991,4 carbon nanotubes (CNTs) have attracted considerable interest because of their outstanding mechanical, electronic,5 and unique surface6,7 properties. * To whom correspondence should be addressed. E-mail: jinoyang@bnu. edu.cn. Fax: 86-10-62799838. † College of Chemistry, Beijing Normal University. ‡ Institute of Cell Biology, Beijing Normal University. § Ghent University. | University Hospital. 10.1021/nl802935s CCC: $40.75 Published on Web 02/27/2009
2009 American Chemical Society
During the past decade, potential biological applications of single-wall (SWNT) and multiwall (MWNT) carbon nanotubes, especially the interactions between CNTs and proteins,5,8-13 have captured much clinical analytical interest. Although most of the studies concern the off-line detection of proteins, these results demonstrate that CNTs cannot only be dispersed in aqueous solutions but also interact with proteins, which provides a potential way to separate and detect proteins using PAGE incorporated with CNTs. This work aims at the development of a novel gel incorporated with CNTs for improving the resolution of a native PAGE in the online detection of human serum proteins. Triton X-100 treated SWNTs and carboxylic MWNTs, which are dispersed in the aqueous solution,8 are introduced into different parts of the gel in the native PAGE system. When brought into the stacking gel, SWNTs act as selective adsorbers to partly adsorb high abundant proteins like immunoglobulin G (IgG), which often hinder the determination of some relatively low-abundance proteins. Furthermore, SWNTs also reduce the background of the electropherogram, resulting in sharper and cleaner proteins bands after CBB-R 250 staining. When added to the resolving gel, c-MWNTs act as a modifier to change the migration speed of some proteins. In addition, an interesting phenomenon was observed in that the separation of specific proteins was enhanced when c-MWNTs were introduced into
Figure 1. Separation of serum proteins by native PAGE with (a) or without (b) SWCNTs present in the stacking gel. (c) Separation of serum proteins after removal of HSA and IgG by native PAGE. Lane 1, 50 µg of IgG. Lanes 2-3, human serum. Human serum diluted 1:10 in 6.67% (v/v) glycerine and 0.05% bromophenol; loading volume: 15 µL.
the specific region of the resolving gel. Last, the method based on the effect of SWNTs in the stacking gel was applied for the detection of sera from patients with liver diseases, and better results were obtained. To study the interaction of carbon nanotubes and proteins in the procedure of electrophoresis, and to establish a method of online high-abundance proteins removal for detecting serum proteins, a novel polyacrylamide gel was manufactured by adding SWNTs to the stacking gel when producing the polyacrylamide gel. As show in Figure 1b, when normal human serum was subjected to native PAGE using a stacking gel without SWNTs, the high abundant protein IgG hinders some other proteins, such as haptoglobin (Hp) and some relatively low-abundance proteins. On the other hand, when electrophoresis was carried out using a stacking gel containing SWNTs, clearer protein bands appeared (Figure 1a). Thus, this method improves resolution because interfering proteins like IgG are adsorbed by the stacking gel and do not migrate into the resolving gel. Furthermore, the electropherogram generally shows sharp, clear bands because of the decreased background. Direct analysis of proteomes especially blood serum by electrophoresis is challenging because of the presence of very high-abundance proteins. To overcome this problem, many approaches have been employed for the depletion of high abundance proteins, such as the affinity-based method, the size-based and the antibody-based depletion method.14 Most of these methods are derived from quite classical approaches largely known in the domain of affinity chromatography, and they do not only offer advantages, but also may result in loss of other low abundance proteins of interest that are associated to the removed carrier proteins such as albumin.1,15-18 The removal of human serum albumin (HSA) and IgG from serum using Aurum Serum Protein Mini Kit (Bio-Rad, U.S.) was comparable to the established method, and the procedure of removal of HSA and IgG was operated according to the instruction manual. As shown in Figure 1c, it could be noticed that during the removal of high abundance proteins, some other species are coadsorbed on the column and therefore also removed. Primary experiments indicated that the interaction between carbon nanotubes and proteins not only exist in solution but Nano Lett., Vol. 9, No. 4, 2009
Figure 2. Separation of serum proteins by native PAGE with or without c-MWCNTs present in the resolving gel. (a) c-MWCNTs present in zone Z of the resolving gel; (b) c-MWCNTs present in zone M of the resolving gel; (c) normal gel.
also in the polyacrlamide gel during the electrophoresis procedure. c-MWNTs had been utilized in capillary electrophoresis because of their exceptional electrical properties.19,20 In order to improve the resolution of native PAGE and to enhance the separation of those serum proteins that were incompletely resolved when detecting with onedimensional (1D) PAGE, c-MWNTs were introduced in the middle region of the resolving gel where some important proteins only incompletely resolved. Native polyacrylamide gel (7.5%, m/v) was used to carry out electrophoresis of human serum, and the results are shown in Figure 2. When polyacrylamide gel was incorporated in the c-MWNTs solution (0.30 × 10-3 g mL-1) in the specific region (Z region of the gel, Figure 2a), four bands (bands 1-4) were determined, whereas only two bands (bands 5 and 6) in the same region with traditional native PAGE (Figure 2c). In order to identify the proteins divided by this method, these four bands (bands 1-4) were excised from the gel. After in-gel digestion, they were analyzed and identified by MS/ MS techniques. Protein identities are listed in Table 1. From the results of the peptide mass fingerprints, band 1 and band 3 are the proteins of apolipoprotein (apo) A-I-1 precursor and apolipoprotein (apo) A-I-2 precursor, band 2 is the protein of Complement C3 precursor, and band 4 the protein of apo A-IV precursor. Complement C3 is mostly synthesized in the liver and correlates with features of the insulin resistance syndrome and predicts the development of diabetes. It is of great diagnostic importance and may indicate progression of atherosclerosis and act as a specific marker of chronic inflammation.21,22 Apo A-I and Apo A-IV are prominent protein constituents of high-density lipoproteins (HDL) and are involved in reverse cholesterol transport to a different extent. They are potent cofactors for the lecithin- cholesterol acyltransferase reaction and bind to cholesterol-loaded cells to promote cellular cholesterol efflux. It is difficult to separate Complement C3 and Apo A-I with native PAGE; apo A-IV is always obscured by Apo A-I23 even in the two-dimensional (2D) electrophoresis system. However, separation of these proteins was achieved by native PAGE with the cited gels. The same protein apo A-I occurred in different positions in the novel gel (Figure 2a, band 1 1321
Figure 3. Effect of different concentrations of c-MWNTs solution on native PAGE. (a) Normal native polyacrylamide gel. (b) Native polyacrylamide gel incorporated with 0.20 mg mL-1 c-MWNTs solution. (c) Native polyacrylamide gel incorporated with 0.30 mg mL-1 c-MWNTs solution. (d) Native polyacrylamide gel incorporated with 0.50 mg mL-1 c-MWNTs solution.
and band 3), indicative of differences in molecular mass (Mr), suggestive of their occurrence as isoforms. The differences between isoforms may represent chemical post-translational modifications or cleavages, given the differences in observed molecular masses.24 Moreover, some other proteins resolved incompletely, besides the proteins mentioned above, when the serum was detected by native PAGE. To investigate whether c-MWNTs would improve the resolution of protein bands in the other region of the gel, the position of the c-MWNTs in the resolving gel was changed when the novel gel was manufactured. Native polyacrylamide gel (7.5%, m/v) was used to carry out electrophoresis of human serum, and the result is shown in Figure 2. It is quite attractive to observe that the protein bands, which cannot be detected by traditional native PAGE, can be also detected after using the novel gel incorporated with c-MWNTs solution. Compared with the normal gel (Figure 2c), separation of some proteins in the marked region was enhanced when polyacrylamide gel was incorporated with c-MWNTs solution (0.20 × 10-3 g mL-1) in the lower part of the resolving gel (the M region of the gel, Figure 2b). Interestingly, from the results it could be observed that when c-MWNTs were introduced into the specific region of the resolving gel with an appropriate concentration (0.30 × 10-3 g mL-1 at the Z region of Figure 2a or 0.20 × 10-3 g mL-1 at the M region of Figure 2b), the resolution of that region at the gel could be improved. In addition, it also became clear that the concentration of c-MWNTs is an important factor for enhancing the separation of those proteins. To further study the effect of concentration of
Figure 4. Electropherogram of normal sera and sera from some patients with liver diseases. Lanes 1-2, normal sera. Lanes 3-4, sera from patients following liver transplantation. Lanes 5-6, sera from patients with hepatic cirrhosis. Lanes 7-8, serum from patient with liver cancer. Lanes 9-10, Hp protein.
c-MWNTs upon separation enhancement of the proteins, the middle region of the gel with c-MWNTs added was selected (the Z region of Figure 2a) and the concentration of c-MWNTs solution in this region was changed when the gel was manufactured. As shown in Figure 3, with c-MWNTs incorporated into the polyacrylamide gel, the migration speed of protein bands 1-4 were changed during the course of electrophoresis, and the change of migration speed of the bands are relevant to the concentration of the c-MWNTs solution. Compared with the normal gel (Figure 3a), no obvious changes were found when the concentration of c-MWNTs solution was set at 0.20 mg mL-1 (Figure 3b). In Figure 3c (0.30 mg mL-1), the separation of Complement C3, Apo A-I, and apo A-IV was achieved. However, the isolation of these proteins cannot be achieved any more when the concentration of c-MWNTs solution was set at 0.50 mg mL-1 (Figure 3d). In the procedure of this experiment, we also found that the c-MWNTs concentration is one of the key factors to achieve a good separation. At optimal and higher c-MWNTs concentrations, each protein shows only a single and narrow band, but at low c-MWNTs concentration however the band tends to broaden; at extremely low c-MWNTs concentrations, the band from a single protein splits into two bands. Hence, the concentration of c-MWNTs and the amount of the protein should be carefully considered during the procedure to carry out a separation. Carbon nanotubes used in the resolving gel were modified by functional groups such as carboxylic and hydroxyl groups, which made the surface of the carbon nanotubes change from hydrophobic to hydrophilic. It is also reported that the
Table 1. MS/MS Results of the Peptide Fragment Searched Employing the PepSea Database Search System Developed by MDS Proteomics spot no. (Figure 2a)
protein name
DB accession
sequence
mass (KDa)
PI
matches
score
1
apolipoprotein A-I precursor Complement C3 precursor apolipoprotein A-I precursor apolipoprotein A-IV precursor
P02647
VSFLSA LEEYTK ILLQGTPVAQ MTEDAIDGER VSFLSA LEEYTK SELTQQLNA LFQDK
30.7780
5.59
24
2520
188.6895
6.35
44
1153
30.7780
5.59
24
1268
45.3993
5.16
31
1238
2 3 4 1322
P01024 P02647 P06727
Nano Lett., Vol. 9, No. 4, 2009
Table 2. Identity of Transferrin (Tf) and Ceruloplasmin (Cp) Marked in Figure 4 protein name
DB accession
sequence
mass (KDa)
PI
matches
score
serotransferrin precursor (transferrin) ceruloplasmin precursor
P02787 P00450
SASDLTWDNLK QSEDSTFYLGER
79.3325 123.0616
7.06 5.56
25 21
1103 389
adsorption process of certain protein on carbon nanotubes is depending on several factors, such as temperature and pH of the environment.13 Hence, the improvement of separation of the divided proteins may be generated by the unique adsorption properties of carbon nanotubes and the special interactions (such as electrostatic interactions, hydrophobic interactions, and π-π interactions) between the c-MWNTs and certain proteins under the condition of the resolving gel. Zhang et al. reported that the carboxylic group of the modified carbon nanomaterials could form covalent bonds with proteins.25 It has been found that some proteins may be encapsulated into the inner space of the CNTs.26 It has also been reported that the binding of proteins to carbon nanotubes is different.27 A mechanic explanation about the resolution needs further study. Because SWNTs were added to the stacking gel reducing background and interference of IgG, the electropherogram looked sharp, and the bands of Cp and Hp appeared clearer. Cp and Hp are very important proteins in human blood; they are members of the acute-phase protein family and have been defined as biomarkers for some liver diseases.28-30 Cp is an R 2-glycoprotein mainly synthesized by hepatocytes, and its serum level is increased during inflammation as well as in various malignancies. However, Cp may be decreased in other disease states like cirrhosis and protein wasting states.30 In these cases, Cp is classically claimed to be decreased in decompensated cirrhosis with hepatic failure30 and may be slightly decreased in other diseases such as fulminant hepatitis.31 Cp level is positively associated with the incidence of cancer.32-34 Hp is an R 2-sialoglycoprotein expressed by a genetic polymorphism as three major phenotypes: 1-1, 2-2, and 2-1, and its gene frequencies show marked geographical differences, such as the phenotypic distribution in the Chinese population indicating that more than 90% of the individuals are Hp 2-1 and Hp 2-2.35 Hp is a hemoglobin binding protein, and its spot is decreased in cirrhotic patients as a result of protein modification.35-38 Ang et al. also reported that the concentrations of serum Hp in hepatocellular carcinoma patients are higher than those in noncancer patients with chronic liver diseases.39 On the basis of the effect of SWNTs in the stacking gel, we applied the method to study the electropherograms of sera from patients with liver diseases so as to find out the relationship between some liver diseases and the level of Hp and Cp. Serum proteins were detected by native PAGE using SWNTs-containing stacking gel, and the Hp phenotypes of these sera were identified by the chemiluminescence imaging method.40 As shown in Figure 4, the Hp phenotype of sera loaded in lanes 1, 4, and 6 is 2-1, and in the other lanes is 2-2. The sera from these patients showed the characteristic response of the levels of Cp and Hp, which can be used for diagnosis of some liver diseases. In patients following liver transplantation (lanes 3-4), Cp and Hp were both markedly decreased. In patients with hepatic cirrhosis Nano Lett., Vol. 9, No. 4, 2009
(lanes 5-6), the Cp level was apparently declined; the Hp level was low in one (lane 6), but its change was not obvious in the other (lane 5). In patients with liver cancer (lanes 7-8), Cp was increased; Hp level in one of the patients was markedly decreased (lane 7), but the change of Hp level in the other was not obvious (lane 8). Hp protein (2-2 and 2-1 forms, Sigma) was loaded in the gel (lanes 9-10) to mark Hp level of sera from the patients. Cp and transferrin (Tf) marked in Figure 4 were identified by the MS/MS techniques, the results being shown in Table 2. The present study describes a novel application of carbon nanotubes. Single-wall and multiwall carbon nanotubes, respectively, introduced into different parts of the gel, have been successfully applied to improve resolution in the detection of human serum with native polyacrylamide gel electrophoresis system. When SWNTs are added to the stacking gel, an online approach to remove IgG while detecting human serum is established, based on the adsorption between SWNTs and IgG. When MWNTs are added to different parts of the resolving gel, the isolation of specific proteins in specific regions of the gel was enhanced, but this could not be achieved with the normal polyacrylamide gel electrophoresis system. This may be due to the change of migration speed of some proteins while MWNTs exist in the gel; the mechanism of separation still needs to be further examined. Last, the method based on the effect of SWNTs in the stacking gel was applied to investigate the serum level of Cp and Hp, which can be used for diagnosis of some liver diseases, and the results being satisfying. In addition, this study also presents a future potential to improve the separation of other proteins or peptides with PAGE or other gel electrophoresis. Acknowledgment. The authors gratefully acknowledge the support from the National Nature Science Foundation of China (20675010). Supporting Information Available: Details of materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ahmed, N.; Barker, G.; Oliva, K.; Garfin, D.; Talmadge, K.; Georgiou, H.; Quinn, M.; Rice, G. Proteomics 2003, 3, 1980–1987. (2) Muratsubaki, H.; Satake, K.; Yamamoto, Y.; Enomoto, K. Anal. Biochem. 2002, 307, 337–340. (3) Sun, H. J.; Pan, Y. C. E. J. Biochem. Biophys. Methods 1999, 39, 143–151. (4) Iijima, S. Nature 1991, 354, 56–58. (5) Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebbesen, T. W.; Mioskowski, C. Angew. Chem., Int. Ed. 1999, 38, 1912–1915. (6) Liu, G. H.; Wang, J. L.; Zhu, Y. F.; Zhang, X. R. Anal. Lett. 2004, 37, 3085–3104. (7) Long, R. Q.; Yang, R. T. J. Am. Chem. Soc. 2001, 123, 2058–2059. (8) Lin, Y.; Taylor, S.; Li, H. P.; Fernando, K. A. S.; Qu, L. W.; Wang, W.; Gu, L. R.; Zhou, B.; Sun, Y. P. J. Mater. Chem. 2004, 14, 527– 541. (9) Azamian, B. R.; Davis, J. J.; Coleman, K. S.; Bagshaw, C. B.; Green, M. L. H. J. Am. Chem. Soc. 2002, 124, 12664–12665. 1323
(10) Shim, M.; Kam, N. W. S.; Chen, R. J.; Li, Y. M.; Dai, H. J. Nano Lett. 2002, 2, 285–288. (11) Huang, W. J.; Taylor, S.; Fu, K.; Lin, Y.; Zhang, D. H.; Hanks, T. W.; Rao, A. M.; Sun, Y. P. Nano Lett. 2002, 2, 311–314. (12) Du, Z.; Yu, Y. L.; Chen, X. W.; Wang, J. H. Chem.sEur. J. 2007, 13, 9679–9685. (13) Valenti, L. E.; Fiorito, P. A.; Garcı´a, C. D.; Giacomelli, C. E. J. Colloid Interface Sci. 2007, 307, 349–356. (14) Kim, M.-R.; Kim, C.-W. J. Chromatogr., B. 2007, 849, 203–210. (15) Granger, J.; Siddiqui, J.; Copeland, S.; Remick, D. Proteomics 2005, 5, 4713–4718. (16) Queloz, P.-A.; Thadikkaran, L.; Crettaz, D.; Rossier, J. S.; Barelli, S.; Tissot, J.-D. Proteomics 2006, 6, 5605–5614. (17) Righetti, P. G.; Castagna, A.; Antonioli, P.; Boschetti, E. Electrophoresis 2005, 26, 297–319. (18) Chromy, B. A.; Gonzales, A. D.; Perkins, J.; Choi, M. W.; Cozett, M. H.; Chang, B. C.; Cozett, C. H.; McCutchen-Maloney, S. L. J. Proteome Res. 2004, 3, 1120–1127. (19) Xiong, X.; Ouyang, J.; Baeyens, W. R. G.; Delanghe, J. R.; Shen, X. M.; Yang, Y. P. Electrophoresis 2006, 27, 3243–3253. (20) Wang, Z. H.; Luo, G. A.; Chen, J. F.; Xiao, S. F.; Wang, Y. M. Electrophoresis 2003, 24, 4181–4188. (21) Somani, R.; Grant, P. J.; Kain, K.; Catto, A. J.; Carter, A. M. Stroke 2006, 37, 2001–2006. (22) Liu, J.; Ouyang, J.; Baeyens, W. R. G.; Delanghe, J. R.; Wang, Z. Z.; Liu, J. X.; Zhang, H. Y. Electrophoresis 2008, 29, 716–725. (23) Schaefer, J. R.; Hackler, R.; Brand, S.; Schwarz, S.; Kleine, T. O.; Steinmetz, A. Clin. Chem. 1995, 41, 76–81. (24) Wang, H.; Clouthier, S. G.; Galchev, V.; Misek, D. E.; Duffner, U.; Ming, C. K.; Zhao, R.; Tra, J.; Omenn, G. S.; Ferrara, J. L. M.; Haash, S. M. Mol. Cell. Proteomics 2005, 4, 618–625. (25) Zhang, M. F.; Yudasaka, M.; Ajima, K.; Miyawaki, J.; Iijima, S. ACS Nano 2007, 1, 265–272.
1324
(26) Shen, J.-W.; Wu, T.; Wang, Q.; Kang, Y. Biomaterials 2008, 29, 3847– 3855. (27) Salvador-Morales, C.; Flahaut, E.; Sim, E.; Sloan, J.; Green, M. L. H.; Sim, R. B. Mol. Immunol. 2006, 43, 193–201. (28) Pieper, R.; Su, Q.; Gatlin, C. L.; Huang, S. T.; Anderson, N. L.; Steiner, S. Proteomics 2003, 3, 422–432. (29) Tao, Q.; Wang, Z. Z.; Zhao, H. P.; Baeyens, W. R. G.; Delanghe, J. R.; Huang, L. Y.; Ouyang, J.; He, D. C.; Zhang, X. H. Proteomics 2007, 7, 3481–3490. (30) Cauza, E.; Dobersberger, T. M.; Palli, C.; Kaserer, K.; Kramer, L.; Ferenci, P. J. Hepatol. 1997, 27, 358–362. (31) Walshe, J. M.; Briggs, J. Lancet 1962, 2, 263–265. (32) Turecky, L.; Kalina, P.; Uhlikova, E.; Namerova, S.; Krizko, J. Klin. Wochenschr. 1984, 62, 187–189. (33) Knekt, P.; Aromaa, A.; Maatela, J.; Rissanen, A.; Hakama, M.; Aaran, R. K.; bNikkari, T.; Hakulinen, T.; Peto, R.; Teppo, L. Br. J. Cancer 1992, 65, 292–296. (34) Yang, M.-H.; Tyan, Y.-C; Jong, S.-B.; Huang, Y.-F; Liao, P.-C.; Wang, M.-C. Anal. Bioanal. Chem. 2007, 388, 637–643. (35) Langlios, M. R.; Delanghe, J. R. Clin. Chem. 1996, 42, 1589–1600. (36) Gravel, P.; Walzer, C.; Aubry, C.; Balant, L. P.; et al. Biochem. Biophys. Res. Commun. 1996, 220, 78–85. (37) Agostoni, A.; Vergani, C.; Stabilini, R.; Marasini, B. Clin. Chim. Acta 1969, 26, 351–355. (38) Tissot, J. D.; Schneider, P.; James, R. W.; Daigneault, R.; et al. Appl. Theor. Electrophor. 1991, 2, 7–12. (39) Ang, I. L.; Poon, T. C. W.; Lai, P. B. S.; Chan, A. T. C.; Ngai, S.-M.; Hui, A. Y.; Johnson, P. J.; Sung, J. J. Y. J. Proteome Res. 2006, 5, 2691–2700. (40) Huang, G. M.; Ouyang, J.; Delanghe, J. R.; Baeyens, W. R. G.; Dai, Z. X. Anal. Chem. 2004, 76, 2997–3004.
NL802935S
Nano Lett., Vol. 9, No. 4, 2009
