Anal. Chem. 2006, 78, 5491-5496
Mixed Monolayer-Protected Gold Nanoclusters as Selective Peptide Extraction Agents for MALDI-MS Analysis Benjamin N. Y. Vanderpuije, Gang Han, Vincent M. Rotello, and Richard W. Vachet*
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
Cationic and anionic nanoparticles selectively target peptides with low and high isoelectric points, respectively. Additionally, their high surface area-to-volume ratios make these nanoparticles (∼2-nm core diameter) very efficient extraction and concentration agents. Upon extraction, the peptide-bound nanoparticles can be analyzed by MALDI-MS to provide highly sensitive detection of the targeted peptides. We demonstrate that MALDI-MS can detect peptide concentrations as low as 500 pM from 250µL solutions using these nanoparticle scaffolds as extraction and concentration agents. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a versatile and effective method for analyzing peptides and proteins;1-4 however, when samples are very complex (e.g., multiprotein digests, cell lysates, tissue extracts, etc.), ionization suppression and adduct formation can limit its effectiveness. Consequently, sample cleanup methods that provide either selective extraction of the desired analytes or selective rejection of interfering species are usually necessary to obtain optimum results. A wide variety of sample preparation approaches have been developed and used to maximize the information content available by MALDI-MS. These preparation techniques fall into two general categoriesson-probe and off-probe purification. On-probe purification techniques simplify the analysis by performing the preconcentration steps on the MALDI target itself, thus reducing sample contamination and analyte losses by minimizing sample handling. Surfaces such as polyethylene5 or poly(vinylidine difluoride)6,7 have been used for general desalting, or more specific surfaces have been employed for targeted purification using surfaceenhanced laser desorption/ionization techniques.8-10 Off-probe techniques include traditional chromatographic separation tech* Corresponding author. E-mail:
[email protected]. Phone: 413545-2733. Fax: 413-545-4490. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (4) Stults, J. T. Curr. Opin. Struct. Biol. 1995, 5, 691-698. (5) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843-848. (6) Strupat, K.; Karas, M.; Hillenkamp, F.; Eckerskorn, C.; Lottspeich, F. Anal. Chem. 1994, 66, 464-470. (7) Vestling, M. M.; Fenselau, C. Anal. Chem. 1994, 66, 471-477. (8) Forde, C. E.; McCutchen-Maloney, S. L. Mass Spectrom. Rev. 2002, 21, 419-439. 10.1021/ac0604181 CCC: $33.50 Published on Web 06/20/2006
© 2006 American Chemical Society
niques and solid-phase extraction methods such as Ziptips. The off-probe techniques are well developed and decouple the extraction and MALDI analysis steps so that both can be independently optimized. Of course, the off-probe approaches require more sample manipulation and increase the potential for contamination or sample loss. An alternative strategy, which provides some of the advantages of both the on- and off-probe purification methods, is to use discrete particles (or beads) to extract and concentrate analytes and then analyze these particles directly by MALDI-MS. Several investigators have used this general approach to purify peptides,11,12 phosphopeptides,13 proteins,14,15 and DNA-binding drugs.16 In these cases, micrometer-sized particles are used for analyte extraction/concentration. Very recently, similar approaches using nanometer-sized particles have been demonstrated.17-22 As extraction agents, nanoparticles (NPs) have significant advantages over micrometer-sized materials. Most notable is a higher surface areato-volume ratio. As particle radius decreases from 1 µm to 10 nm, for example, the surface area-to-volume ratio increases 100-fold. This increase translates into much greater extraction capacity, and NPs have great potential in this regard. Indeed, several researchers have recently used the inherent extraction efficiency of NPs in conjunction with MALDI-MS analysis. Chen and coworkers have demonstrated that gold NPs with negatively charged (9) Issaq, H. J.; Conrads, T. P.; Prieto, D. A.; Tirumalai, R.; Veenstra, T. D. Anal. Chem. 2003, 75, 149A-155A. (10) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23, 34-44. (11) Gevaert, K.; Demol, H.; Puype, M.; Broekaert, D.; De Boeck, S.; Houthaeve, T.; Vandekerckhove, J. Electrophoresis 1997, 18, 2950-2960. (12) Gevaert, K.; Demol, H.; Sklyarova, T.; Vandekerckhove, J.; Houthaeve, T. Electrophoresis 1998, 19, 909-917. (13) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borchers, C. H. Anal. Chem. 2002, 74, 3429-3433. (14) Yaneva, M.; Tempst, P. Anal. Chem. 2003, 75, 6437-6448. (15) Villanueva, J.; Phillip, J.; Entenberg, D.; Chaparr, C.; Tanwar, M.; Holland, E.; Tempst, P. Anal. Chem. 2004, 76, 1560-1570. (16) Schlosser, G.; Vekey, K.; Malorni, A.; Pocsfalvi, G. Rapid Commun. Mass Spectrom. 2005, 19, 3307-3314. (17) Teng, C.-H.; Ho, K.-C.; Lin, Y.-S.; Chen, Y.-C. Anal. Chem. 2004, 76, 43374342. (18) Chen, C.-T.; Chen, Y.-C. Anal. Chem. 2005, 77, 5912-5919. (19) Sudhir, P.-R.; Wu, H.-F.; Zhou, Z.-C. Anal. Chem. 2005, 77, 7380-7385. (20) Turney, K.; Drake, T. J.; Smith, J. E.; Tan, W.; Harrison, W. W. Rapid Commun. Mass Spectrom. 2004, 18, 2367-2374. (21) 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. (22) Zhang, Y.; Wang, X.; Shan, W.; Wu, B.; Fan, H.; Yu, X.; Tang, Y.; Yang, P. Angew. Chem., Int. Ed. 2005, 44, 615-617.
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surfaces can trap and concentrate positively charged peptides from 100 nM solutions17 while TiO2-coated Fe3O4 NPs can be used to selectively concentrate phosphopeptides at 500 pM concentrations.18 Gold NPs dissolved in toluene have been used as part of a liquid-liquid microextraction system to concentrate negatively charged peptides present at micromolar concentrations in urine samples.19 Other NP materials such as silica,20 diamond,21 and zeolites22 have also been used to concentrate peptides from very dilute solutions. In addition to high surface area-to-volume ratios, some NPs offer the ability to build materials with “bottom-up” design control. This feature enables the creation of particles with surface functionality that can selectively recognize a particular class of compound.23-31 In particular, monolayer-protected gold clusters (MPCs), which are core/shell NPs, provide many desirable attributes for the creation of selective extraction agents. MPCs can be formed via a one-pot synthesis.32 Their core size can be varied from
