Anal. Chem. 2008, 80, 2583-2589
Modified Silver Nanoparticle as a Hydrophobic Affinity Probe for Analysis of Peptides and Proteins in Biological Samples by Using Liquid-Liquid Microextraction Coupled to AP-MALDI-Ion Trap and MALDI-TOF Mass Spectrometry Kamlesh Shrivas and Hui-Fen Wu*
Department of Chemistry, National Sun Yat-Sen University, Taiwan, and National Sun Yat-sen UniversitysKaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan
A new approach of using modified silver nanoparticles (AgNPs) in toluene as hydrophobic affinity probes for the separation and preconcentration of peptides and proteins in biological samples prior to atmospheric pressurematrix assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry and matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry has been successfully demonstrated. To our best knowledge, for the first time, the modified AgNPs with hydrophobic ligands, such as dodecanethiol (DT) and octadecanethiol (OT) in toluene, were used for the liquidliquid microextraction (LLME) of peptides and proteins through the hydrophobic interactions. In the present investigation, gramicidin was chosen as a model compound to assess the hydrophobic extraction with the modified AgNPs. The optimum extraction efficiency of gramicidine was observed at pH 7.0 for 1.5 h of extraction time with 7% addition of salt. Compared to the conventional use of AP-MALDI-MS, a 266-388-fold improvement in the limit of detection (LOD) for gramicidin was obtained in urine and plasma samples. The lowest concentration of gramicidin that was detected by using modified AgNPs in urine and plasma samples was 0.13 and 0.16 µM, respectively. Furthermore, the proposed method was demonstrated for the extraction of other long chain proteins, like myoglobin, ubiquitin, and bovine serum albumin, in a sample solution by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The major feature of the newly synthesized modified AgNPs was that the target species could be efficiently separated and preconcentrated without sample loss prior to MALDI-MS detection for the sensitive and effective analysis of peptides and proteins in biological samples. Matrix assisted laser desorption/ionization (MALDI) is a laser based soft ionization technique proven to be one of the most powerful ionization techniques for mass spectrometric analysis and investigation of peptides, proteins, nucleic acids, pharmaceu* Corresponding author. Phone: 886-7-5252000-3955. Fax: 886-7-525-3908. E-mail:
[email protected]. 10.1021/ac702309w CCC: $40.75 Published on Web 03/07/2008
© 2008 American Chemical Society
ticals, bacterial characterization, and imaging studies.1-8 The sample cleanup is the most important step for isolation of interfering substances from complex biological samples prior to instrumental analysis.5,6,8 Several surface derivatization procedures for sample preparations have been designed to extract and concentrate the analyte through hydrophobic interaction,9,10 ionic interaction,11,12 or immunoaffinity.13,14 Recently, nanoparticles have been recognized as effective probes in the separation of analytes such as phosphopeptides, antigens, oligonucleotides, proteins, carbohydrates, and bacteria, etc.15-22 Chen et al.23 reported the use of negatively functionalized (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Lin, P. C.; Chou, P. H.; Chen, S. H.; Liao, K. L.; Wang, K. Y.; Chen. Y. J.; Lin, C. C. Small 2006, 4, 485-489. (3) Domon, B.; Aebersold, R. Science 2006, 312, 212-217. (4) Berkenkamp, S.; Kirpekav, F.; Hillenkamp, F. Science 1998, 281, 260262. (5) Wu, J.; Chatman, K.; Harris, K.; Siuzdak, G.; Ho, K. C.; Tsai, P. J.; Lin, Y. S.; Chen, Y. C. Anal. Chem. 2004, 76, 7162-7168. (6) Shrivas, K.; Wu, H. F. Rapid Commun. Mass Spectrom. 2007, 21, 31033108. (7) Bunch, J.; Clench, M. R.; Richards, D. S. Rapid Commun. Mass Spectrom. 2004, 18, 3051-3060. (8) Lin, Y. S.; Tsai, P. J.; Weng, M. F.; Chen, Y. C. Anal. Chem. 2005, 77, 1753-1760. (9) Brockman, A. H.; Shah, N. N.; Orlando, R. J. Mass Spectrom. 1998, 33, 1141-1147. (10) Brockman, A. H.; Dodd, B. S.; Orlando, R. Anal. Chem. 1997, 69, 47164720. (11) Xu, Y.; Watson, T.; Bruening, M. L. Anal. Chem. 2003, 75, 185-190 (12) Warren, M. E.; Brockman, A. H.; Orlando, R. Anal. Chem. 1998, 70, 37573761. (13) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (14) Liang, X.; Lubman, D. M.; Rossi, D. T.; Nordblom, G. D.; Barksdale, C. M. Anal. Chem. 1998, 70, 498-503. (15) Lin, H. Y.; Chen, C. T.; Chen, Y. C. Anal. Chem. 2006, 78, 6873-6878. (16) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919. (17) Chou, P. H.; Chen, S. H.; Liao, H. K.; Lin, P. C.; Her, G. R.; Lai, A. C. Y.; Chen, J. H.; Lin, C. C.; Chen, Y. J. Anal. Chem. 2005, 77, 5990-5997. (18) Flad, T.; Schiestel, T.; Brunner, H.; Tolson, J.; Ouyang, Q.; Pawelec, G.; Mueller, G. A.; Mueller, C. A.; Tovar, G. E. M.; Beck, H. J. Immunol. Methods 2003, 283, 205-213. (19) Maier, M.; Fritz, H.; Gerster, M.; Schewitz, J.; Bayer, E. Anal. Chem. 1998, 70, 2197-2204. (20) Kong, X.; Huang, L. C. L.; Liau, S. C. V.; Han, C. C.; Chang, H. C. Anal. Chem. 2005, 77, 4273-4277. (21) Chen, W. Y.; Wang, L. S.; Chiu, H. T.; Chen, Y. C. J. Am. Soc. Mass Spectrom. 2004, 15, 1629-1635.
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008 2583
magnetic gold nanoparticles as affinity probes in trapping positively charged species from aqueous solutions through the electrostatic force of attractions. Chang et al.20,24 demonstrated the use of carboxylated diamond nanoparticles in capturing target proteins and oligonucleotides from the sample solution by means of hydrophilic and hydrophobic forces. Chou et al.17 also developed magnetic nanoparticles coated with cross linker (N-hydroxysuccinimide ester) as affinity probes to isolate and preconcentrate target antigens from biological media through covalent bonding. However, these techniques require tedious, laborious, and complex sample preparation processes prior to MALDI-MS analysis. Washing of nanoparticles after extraction may also cause analyte loss and chance of interference of matrixes from samples. Direct analysis of analytes with nanoparticles in the organic solvent limits the analyte loss and provides the opportunity to concentrate peptides into a volume of organic solvent. We previously25 reported the use of modified gold nanoparticles assisted with single drop microextraction (SDME) in the separation and preconcentration of hydrophilic peptides from sample solutions before MALDI-MS analysis. This approach is simple, and samples can be directly deposited on target plates for MALDI-MS analysis without sample loss. As the discussion pertains to MALDI-MS, hydrophilic peptides are easily analyzed using the modified nanoparticles or other means of sample preparation techniques. However, the analysis of hydrophobic peptides is more difficult due to limited solubility in aqueous solutions. In the present study, liquid-liquid microextraction (LLME) based modified AgNPs have been employed for the extraction of a hydrophobic peptide (gramicidin) from biological samples through hydrophobic interactions. The hydrophobic interaction, which is a force driving hydrophobic molecules (segments) away from the polar environment and into the nonpolar domain, provides significant characteristics in biorecognition systems. Besides, in reversed phase chromatography, the separation of the analyte is based on hydrophobic interactions with the alkyl chain of the stationary phase to the nonpolar endgroup of the analytes. In general, an analyte with a longer alkyl chain results in a longer retention time because it increases the molecule’s hydrophobicity.26-28 The thermodynamic studies are also described for the hydrophobic interaction of polypeptide with nonpolar ligands.29,30 However, Orlando et al. demonstrated the hydrophobic interaction between the self-assembled monolayer of octadecyl mercaptan and octadecanethiol (C18) with polypeptide in the isolation and concentration of analytes for MALDI-MS (22) Su, C. L.; Tseng, W. L. Anal. Chem. 2007, 79, 1626-1633. (23) Teng, C. H.; Ho, K. C.; Lin, Y. S.; Chen, Y. C. Anal. Chem. 2004, 76, 43374342. (24) Kong, X. L.; Huang, L. C. L.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang, H. C. Anal. Chem. 2005, 77, 259-265. (25) Sudhir, P. R.; Wu, H. F.; Zhou, Z. C. Anal. Chem. 2005, 77, 73807385. (26) Reverse phase HPLC basics for LC-MS, http://www.ionsource.com/ tutorial/chromato graphy/rphplc.htm. (27) Dias-Cabral, A. C.; Ferreira, A. S.; Phillips, J.; Queiroz, J. A.; Pinto, N. G. Biomed. Chromatogr. 2005, 19, 606-616. (28) Queiroz, J. A.; Tomaz, C. T.; Cabral, J. M. S. J. Biotechnol. 2001, 87, 143159. (29) Hearn, M. T. W.; Zhao, G. Anal. Chem. 1999, 71, 4874-4885. (30) Boysen, R. I.; Wang, Y.; Keah, H. H.; Hearn, M. T. W. Biophys. Chem. 1999, 77, 79-97.
2584
Analytical Chemistry, Vol. 80, No. 7, April 1, 2008
analysis.12,13 Harrsion et al.31 presented aptamer modified nanoparticles in the hydrophobic extraction of peptides from biological samples. Among noble metals, silver nanoparticles are used in the surface enhanced Raman scattering and surface plasmon resonance studies due to their distinct physical, chemical, and optoelectronic properties.32,33 In addition, AgNPs have shown antibacterial activities against some bacteria.34 For these reasons, and based upon previous work regarding the interactions of noble metal nanoparticles with biomolecules,22,23,25 we decided to develop AgNPs based affinity probes in the sample preparation for peptides and proteins. The surface of AgNPs were modified by the dodecanethiol (DT) and octadecanethiol (OT) containing hydrophobic ligands in toluene and used for the LLME of gramicidin, prior to AP-MALDI-MS analysis. The governable factors, including extraction time, pH, salts, temperature, and alkyl chain length, were optimized for better extraction and sensitivity of gramicidin. The applicability of the proposed method has been illustrated in the determination of gramicidin from urine and plasma samples. Further, the feasibility of the proposed method was extended for the analysis of larger proteins including myoglobin, ubiquitin, and BSA in sample solutions by using MALDI-TOF-MS. EXPERIMENTAL SECTION Reagents and Materials. All reagents used were of analytical grade. Toluene and silver nitrate were obtained from Mallinckrodt (Paris, KY). Sodium borohydride was purchased from Fluka (Steinheime, Germany). R-Cyano-4-hydroxycinnamic acid, (CHCA), sinapinic acid (SA), gramicidin D, horse heart myoglobin (Mb), bovine serum albumin (BSA), bovine red blood cells ubiquitin (Ub), dodecanethiol, and octadecanethiol were purchased from Sigma-Aldrich (St. Louis, MO). Gramicidin D consists of three major gramicidin species, A, B, and C, and have the amino acid sequence formyl-L-Val1-D-Gly2-L-Ala3-D-Leu4-L-Ala5-D-Val6-L-Val7-DVal8-L-Trp9-D-Leu10-L-Xxx11-D-Leu12-L-Trp13-D-Leu14-L-Trp15-ethanolamine, where Xxx11 is Trp in gramicidin A (GA), Phe in gramicidin B (GB), and Tyr in gramicidin C (GC). The Vortex Agitator (VM 2000, DigiSystem Laboratory, Taiwan) was used for agitation of the samples. Standard Solutions. A stock standard solution of gramicidin (0.60 mM) was prepared by dissolving 1 mg of substance in 1 mL of methanol. A 5 µM stock standard solution of myoglobin, ubiquitin, and BSA were prepared separately in a 50 mM NH4HCO3 solution. Working standard solutions were prepared by the dilution of stock solution with deionized water. Matrix solutions of 0.026 M CHCA and 0.089 M SA were prepared separately in a 2:1 mixture of ACN-H2O which contained 0.1% trifluoroacetic acid (TFA). Safety steps were maintained during the handling of organic solvents and chemicals. (31) Turney, K.; Drake, T. J.; Smith, J. E.; Tan, W.; Harrsion, W. W. Rapid Commun. Mass Spectrom. 2004, 18, 2367-2374. (32) Kundu, S.; Mandal, M.; Ghosh, S. K.; Pal, T. J. Photochem. Photobiol., A: Chem. 2004, 162, 625-632. (33) Haes, A. J.; Van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 1059610604. (34) Sondi, I.; Salopek-Sondi, B. J. Colloid Interface Sci. 2004, 275, 177-182.
Figure 1. TEM images of modified AgNPs with (a) dodecanethiol and (b) octadecanethiol ligands.
Preparation of Modified Silver Nanoparticles in Toluene. Hydrosols of silver were prepared by the reduction of AgNO3 solution with NaBH4 as described elsewhere.35 Briefly, 0.5 mL of 0.01 M NaBH4 was added dropwise to 100 mL of 0.29 mM AgNO3 solution for 3 h. The solution turns light yellow after addition of all the NaBH4 solution. The prepared hydrosols of silver in aqueous solution were added to the toluene solution containing 1.66 mM dodecanethiol (DT) or octadecanethiol (OT), after dropwise addition of 1.0 mL of concentrated HCl to the vigorously stirred solution for 1 h. Hydrosols of silver were transferred to the organic phase, as evidenced by the color change resulting in modified AgNPs with DT and OT in toluene. Characterization of Modified AgNPs. UV-Visible. The UVvis spectra of modified AgNPs were recorded using a double beam UV-vis spectrophotometer (Hitachi, Japan) between 300 and 800 nm. (Supporting Information, Figure S1). The UV absorption measurements revealed that the maximum wavelengths were recorded at 425 nm; these values are consistent with the expected sizes of the modified AgNPs. TEM. The size of the AgNPs was further confirmed withtransmission electron microscopy (TEM) (JEOL, Tokyo, Japan) at an accelerating voltage of 200 kV. An amount of 0.5 µL of toluene containing modified AgNPs was deposited on the copper grid and vacuum-dried. The average size of the modified AgNPs with DT and OT obtained were
