Solid-Phase Extraction and Elution on Diamond (SPEED): A

Acceleration of Microwave-Assisted Enzymatic Digestion Reactions by Magnetite Beads. .... Application of Nanodiamonds in Biomolecular Mass Spectrometr...
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Anal. Chem. 2006, 78, 4228-4234

Solid-Phase Extraction and Elution on Diamond (SPEED): A Fast and General Platform for Proteome Analysis with Mass Spectrometry Wei-Hao Chen,†,‡ Sheng-Chung Lee,‡,§ Sahadevan Sabu,† Huei-Chun Fang,† Shu-Chien Chung,† Chau-Chung Han,*,†,| and Huan-Cheng Chang*,†,|

Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-116, Taipei 106, Taiwan, R.O.C., Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan, R.O.C., and Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan, R.O.C.

This paper presents a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purification and concentration of intact proteins and their enzymatic digests for ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry analysis without prior removal of the adsorbent. One-pot work flow involving reduction of disulfide bonds, protection of free cysteine residues, washing off residual chemicals, and proteolytic digestion of adsorbed proteins can be performed directly on the particles. The utility and versatility of this protein workup platform were demonstrated with liquid chromatography-electrospray ionization tandem mass spectrometry in proteome analysis of human urine. The proteome analysis of each urine sample can be completed in 8 h. An important facet of contemporary bioanalysis is to develop techniques and methodologies that expedite the process of proteomics research. Mass spectrometry has emerged as one of these core techniques,1 and it is a promising diagnostic tool for identifying biomarkers related to human diseases.2,3 A widely accepted approach in this field is to separate protein components from clinical specimens (such as humoral fluids) by one- or twodimensional gel electrophoresis, followed by in-gel proteolytic digestion, and, finally, mass spectrometric identification.4-10 Al* To whom correspondence should be addressed. E-mail: cchan@ pub.iams.sinica.edu.tw; [email protected]. † Institute of Atomic and Molecular Sciences, Academia Sinica. ‡ National Taiwan University. § Institute of Biological Chemistry, Academia Sinica. | Genomics Research Center, Academia Sinica. (1) Godovac-Zimmermann, J.; Brown, L. R. Mass Spectrom. Rev. 2001, 20, 1-57. (2) Wittke, S.; Mischak, H.; Walden, M.; Kolch, W.; Ra¨dler, T.; Wiedemann, K. Electrophoresis 2005, 26, 1476-1487. (3) Chalmers, M. J.; Mackay, C. L.; Hendrickson, C. L.; Wittke, S.; Walden, M.; Mischak, H.; Fliser, D.; Just, I.; Marshall, A. G. Anal. Chem. 2005, 77, 7163-7171. (4) Samyn, B.; Sergeant, K.; Castanheira, P.; Faro, C.; Van Beeumen, J. Nat. Methods 2005, 2, 193-200. (5) Oh, J.; Pyo, J.-H.; Jo, E.-H.; Hwang, S.-I.; Kang, S.-C.; Jung, J.-H.; Park, E.K.; Kim, S.-Y.; Choi, J.-Y.; Lim, J. Proteomics 2004, 4, 3485-3497.

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ternatively, the entire protein mixture can be digested either enzymatically or chemically, and the resulting complicated peptide mixture is submitted to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.11-13 In the latter approach, removal of physiological and processing contaminants such as salts, detergents, and chemical reagents is usually required to prevent adverse interference of these compounds in proteolysis. Moreover, concentration of diluted sample solutions is needed prior to ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoriese (SDS-PAGE) and MS analyses. However, the commonly used procedures for protein purification and concentration such as trichloroacetic acid (TCA) precipitation and dialysis with a molecular size cutoff membrane are generally timeconsuming, incomplete in removing contaminants, and plagued with high sample loss to membranes or the walls of containers.5,14 Furthermore, the presence of high-abundance proteins often spoils the MS identification of minute components, and therefore, additional steps for depletion of these abundant proteins have to be undertaken.15-19 (6) Wattiez, R.; Falmagne, P. J. Chromatogr., B 2005, 815, 169-178. (7) Finehout, E. J.; Franck, Z.; Lee, K. H. Electrophoresis 2004, 25, 25642575. (8) Pieper, R.; Gatlin, C. L.; Makusky, A. J.; Russo, P. S.; Schatz, C. R.; Miller, S. S.; Su, Q.; McGrath, A. M.; Estock, M. A.; Parmar, P. P.; Zhao, M.; Huang, S.-T.; Zhou, J.; Wang, F.; Esquer-Blasco, R.; Anderson, N. L.; Taylor, J.; Steiner, S. Proteomics 2003, 3, 1345-1364. (9) Pieper, R.; Gatlin, C. L.; McGrath, A. M.; Makusky, A. J.; Mondal, M.; Seonarain, M.; Field, E.; Schatz, C. R.; Estock, M. A.; Ahmed, N.; Anderson, N. G.;. Steiner, S. Proteomics 2004, 4, 1159-1174. (10) Tantipaiboonwong, P.; Sinchaikul, S.; Sriyam, S.; Phutrakul, S.; Chen, S.-T. Proteomics 2005, 5, 1140-1149. (11) He, P.; He, H.-Z.; Dai, J.; Wang, Y.; Sheng, Q.-H.; Zhou, L.-P.; Zhang, Z.-S.; Sun, Y.-L.; Liu, F.; Wang, K.; Zhang, J.-S.; Wang, H.-X.; Song, Z.-M.; Zhang, H.-R.; Zeng, R.; Zhao, X.-H. Proteomics 2005, 5, 3442-3453. (12) Pang, J. X.; Ginanni, N.; Dongre, A. R.; Hefta, S. A.; Opitek, G. J. J. Proteome Res. 2002, 1, 161-169. (13) Spahr, C. S.; Davis, M. T.; McGinley, M. D.; Robinson, J. H.; Bures, E. J.; Beierle, J.; Mort, J.; Courchesne, P. L.; Chen, K.; Wahl, R. C.; Yu, W.; Luethy, R.; Patterson, S. D. Proteomics 2001, 1, 93-107. (14) Doucette, A.; Craft, D.; Li, L. Anal. Chem. 2000, 72, 3355-3362. (15) Bjo ¨rhall, K.; Miliotis, T.; Davidsson, P. Proteomics 2005, 5, 307-317. (16) Chen, Y.-Y.; Lin, S.-Y.; Yeh, Y.-Y.; Hsiao, H.-H.; Wu, C.-Y.; Chen, S.-T.; Wang, A. H.-J. Electrophoresis 2005, 26, 2117-2127. (17) Cho, S. Y.; Lee, E.-Y.; Lee, J. S.; Kim, H.-Y.; Park, J. M.; Kwon, M.-S.; Park, Y.-K.; Lee, H.-J.; Kang, M.-J.; Kim, J. Y.; Yoo, J. S.; Park, S. J.; Cho, J. W.; Kim, H.-S.; Paik, Y. K. Proteomics 2005, 5, 3386-3396. 10.1021/ac052085y CCC: $33.50

© 2006 American Chemical Society Published on Web 05/16/2006

Solid-phase extraction (SPE) is a popular solution to overcoming these difficulties. A number of attempts have been made previously14,20-23 to use surface-modified substrates to extract proteins and peptides, either specifically or nonspecifically, from highly dilute and contaminated sample solution, followed by enzymatic digestion of the adsorbed molecules with or without an elution step. Hydrophobic particles14 and chemically modified matrixes (such as diamond powders20 and polymeric beads21) are typical adsorbents used for such purpose. However, the extraction capabilities of hydrophobic surfaces have been shown to deteriorate greatly in solutions containing more than 0.02% SDS.14 MS analysis of trypsinized peptides from these extracted proteins revealed less information than proteins concentrated from SDSfree solutions. Moreover, a significant fraction of peptide fragments after the tryptic digestion was trapped in the porous material, resulting in low peptide recovery efficiency. Here, we present a simple but effective solid-phase extraction and elution platform for systematic proteome analysis of humeral fluids and, potentially, as a tool for disease biomarker discovery. The platform, solid-phase extraction and elution on diamond, which we refer to as SPEED, is established based on our previous studies of surface-functionalized nanodiamonds.24,25 We showed that diamond nanocrystallites, after being treated in strong oxidative acids, have exceptionally high affinity for proteins (and also some peptides) due to the interplay of electrostatic forces, hydrogen bonding and hydrophobic interactions between adsorbate and surface. The smallness, inertness, and optical transparency of the nanocrystallites allowed direct mass analysis of the diamond-bound proteins with matrix-assisted laser desorption/ ionization (MALDI)-MS without prior removal of the adsorbent.25 In this note, we demonstrate further that these surface-functionalized nanodiamonds can be used as SPE supports to extract not only proteins but also protein digests from dilute solutions containing high concentrations of urea, salts, and detergents. Nondiscriminative extraction can be easily achieved at acidic pH. Furthermore, proteins bound to nanodiamonds can be analyzed facilely by SDS-PAGE with electrophoresis buffers containing 1% SDS as the eluent. Chemical processing (i.e., disulfide bond reduction, alkylation of free cysteines, and proteolytic digestion) can be conducted directly on the particle. The resulting peptide fragments can be either freed from or attached to the diamond particles, depending on the ionization technique (electrospray ionization, ESI, or MALDI) to be used in ensuing MS analysis. A real-world application of this sample manipulation platform is demonstrated with proteome-wide analysis of human urine. By (18) Ogata, Y.; Charlesworth, M. C.; Muddiman, D. C. J. Proteome Res. 2005, 4, 837-845. (19) Zolotarjova, N.; Martosella, J.; Nicol, G.; Bailey, J.; Boyes, B. E.; Barrett, W. C. Proteomics 2005, 5, 3304-3313. (20) Xu, J.; Reilly, J. P. Proceedings of INTERTECH 2003, Industrial Diamond Association of America, Columbus, OH, 2003. (21) Janecki, D. J.; Broshears, W. C.; Reilly, J. P. Anal. Chem. 2004, 76, 66436650. (22) Chen, Y.-J.; Chen, S.-H.; Chien, Y.-Y.; Chang, Y.-W.; Liao, H.-K.; Chang, C.-Y.; Jan, M.-D.; Wang, K.-T.; Lin, C.-C. ChemBioChem 2005, 6, 11691173. (23) Jessani, N.; Niessen, S.; Wei, B. Q.; Nicolau, M.; Humphrey, M.; Ji, Y.; Han, W.; Noh, D.-Y.; Yates, J. R.; Jeffrey, S. S.; Cravatt, B. F. Nat. Methods 2005, 2, 691-697. (24) Huang, L.-C. L.; Chang, H.-C. Langmuir 2004, 20, 5879-5884. (25) 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.

prefractionating urinary proteins with nanodiamonds at different pHs for LC-ESI-MS/MS, we identified 125 proteins in urine samples collected from 10 healthy males and 10 healthy females without complicated and time-consuming procedures. EXPERIMENTAL SECTION Chemical and Materials. Analytical grade methanol and acetonitrile were from Baker. Bovine heart cytochrome c, bovine serum albumin, horse heart myoglobin, glycine, Tris, glycerol, β-mercaptoethanol, bromophenol blue, Triton X-100, sodium citrate, CAPS, ammonium bicarbonate, and 2,5-dihydroxybenzoic acid (DHB) were from Sigma. Acetic acid, formic acid, 4-vinylpyridine, trichloroacetic acid, and trifluoroacetic acid (TFA) were from Acros Organics. Sodium dodecyl sulfate, urea, CHAPS, and coomasie brilliant blue R-250 were from Bio-Rad. Sequence grade trypsin for protein digestion was from Promega. Abrasive diamond powders of sizes in the range of 100 nm were obtained from Kay Industrial Diamond. Deionized (DI) water was prepared with an Elga UHQ water purification system. Protein Extraction and Elution. Diamond nanocrystallites were purified and surface-functionalized with strong oxidative acid treatments, as previously described.24,25 The acid-treated nanodiamonds were then suspended in DI water or buffer solution at a nominal concentration of 1 mg/mL. To evaluate the protein adsorbability of the adsorbent, 20 µL of the nanodiamond suspension was pipetted immediately after homogenization by vortexing and added to 500 µL of protein solutions of known concentrations. The mass ratio of the total proteins to the nanodiamonds was kept at a level of 1:50 (unless otherwise stated),26 below saturation of the adsorbents.24,25 After incubation for 5 min at room temperature, the protein-diamond mixture was centrifuged at 12 000 rpm for 3 min. The supernatant was collected, while the precipitated nanodiamonds were washed with 500 µL of the corresponding buffer used for protein adsorption. Prior to PAGE and MS analyses, the protein-loaded nanodiamonds were additionally washed with acetonitrile to remove residual contaminants. In the case of urinary protein extraction, the pH values of the sample solutions were first adjusted by adding 50 µL of 0.5 M sodium citrate (pH 3.0), Tris-HCl (pH 7.0) or CAPS (pH 11.0) to 500 µL of urine. After elimination of solid components by centrifugation, 100 µg of nanodiamonds was added to the supernatant to extract the proteins. Protein Modification and Digestion. On-particle digestion was conducted by adding protein-loaded nanodiamonds to 50 µL of 25 mM NH4HCO3 solution containing 5 ng of trypsin (or 50 ng in urinary protein digestion). After incubation at 50 °C for 1 h, the mixture was dried using a centrifugal vacuum evaporator and stored at -20 °C until use. In-solution digestion was performed in parallel as a control experiment without adding nanodiamonds to the protein solution. To modify proteins containing intramolecular disulfide bonds, protein-loaded nanodiamonds were first incubated in 50 µL of a 2% β-mercaptoethanol/25 mM NH4HCO3 stock solution for 10 min. The same volume of the solution composed of 50% acetonitrile, 10% 4-vinylpyridine, and 25 mM (26) A 1:20 mass ratio of the total proteins to the nanodiamonds was found sufficient for effective protein adsorption, but increasing the diamond dosage to a level of 50 times the protein mass facilitates high-throughput operations due to reduced relative loss in collection of nanodiamonds after centrifugation.

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NH4HCO3 was then added for cysteine alkylation. After incubation for 10 min, the protein-diamond mixture was washed with 500 µL of acetonitrile to remove residual reducing and alkylating reagents prior to tryptic digestion at room temperature. In-gel digestion27 and TCA precipitation16 were conducted following the standard protocols in the literature. Specifically in TCA precipitation, 50 µL of TCA was added to 450 µL of sample solution to a final concentration of 10%. After incubation at -20 °C for 90 min, the mixture was centrifuged at 15 000 rpm at 4 °C for 20 min. The precipitate collected was redispersed in 1 mL of ice-cold acetone, after which the mixture was incubated on ice for 15 min, centrifuged as above, and lyophilized for subsequent SDS-PAGE analysis. SPEED-SDS-PAGE. Protein-loaded nanodiamonds were mixed with sample buffers (75 mM Tris-HCl, 2.4% SDS, 5% β-mercaptoethanol, 0.05% bromophenol blue, and 1 M urea at pH 6.8) and incubated at 95 °C for 5 min. The mixture was then electrophoresized through a 15% Tris-glycine gel using a Mini Protean 3 cell (Bio-Rad) at a constant current of 20 mA per slab gel.28 Silver staining and coomasie blue staining were performed to visualize the separated proteins. SPEED-MALDI-MS. Dried peptide-nanodiamond mixtures from tryptic digestion were suspended in 5 µL of 100 mg/mL DHB prepared with 70% acetonitrile and 0.1% TFA. After centrifugation to separate nanodiamonds, 0.5 µL of the supernatant was deposited onto a stainless steel target for MALDI-MS analysis with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (APEX-IV, Bruker-Daltonics) equipped with a 7-T magnet and a vacuum MALDI source (Scout-100). SPEED-LC-ESI-MS/MS. On-diamond-digested peptide fragments were analyzed using a quadrupole ion trap mass spectrometer (Esquire 3000 plus, Bruker-Daltonics) interfaced with a solvent delivery system (UltiMate, LC-Packings) and an autosampler (Famos, LC-Packings). A 150 × 0.5 mm C18 column (Zorbax 300SB, Agilent) served to separate the peptide fragments. The mobile phases for liquid chromatography consisted of 0.05% formic acid in water and 0.05% formic acid in acetonitrile. The peptides were eluted at a flow rate of 1 µL/min with an acetonitrile gradient from 15 to 65% in 60 min. The acquired mass spectra were analyzed and processed using the Data Analysis and BioTools software packages. The in-house version of the Mascot search engine (Matrix Science) was used for comparison of the obtained MS/MS spectra with the theoretical spectra derived from the protein sequence database at the National Center for Biotechnology Information. RESULTS AND DISCUSSION The core materials used in the SPEED platform are diamond nanocrystallites with a nominal size of 100 nm. These crystallites are commercially available mainly for surface-finishing applications. Upon purification in strong oxidative acids, their surfaces are automatically functionalized with carboxyl, carbonyl, and a variety of oxygen-containing groups. Although the composition of these functional groups has not yet been fully characterized, such nanodiamonds show exceptionally high affinity for proteins in highly dilute and severely contaminated solutions.24,25 However, (27) Tsay, Y. G.; Wang, Y. H.; Chiu, C. M.; Shen, B. J.; Lee, S. C. Anal. Biochem. 2000, 287, 55-64. (28) Laemmli, U. K. Nature 1970, 227, 680-685.

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Figure 1. SDS-PAGE analysis of myoglobin extracted from solutions containing SDS using nanodiamonds as the solid-phase extraction supports. In this experiment, 1 µg of horse heart myoglobin dissolved in 500 µL of solution (deionized water or 40% methanol) containing various concentrations of the interfering reagents was extracted with 20 µg of nanodiamonds. Control experiments: C1, direct loading of 1 µg of myoglobin; C2, 1 µg of myoglobin extracted with 20 µg of nanodiamonds without SDS.

Figure 2. SDS-PAGE analysis of myoglobin extracted from solutions containing (a) urea and (b) acetonitrile using nanodiamonds as the solid-phase extraction supports. The experimental conditions used are same as those described in Figure 1.

further experimentation indicated that the presence of ionic detergents such as SDS in solution would substantially reduce the affinity of proteins for nanodiamonds. Figure 1 shows a result of the SDS-PAGE analysis using myoglobin as a model protein, where no sign of protein adsorption was detected when SDS concentration in the sample solution is higher than 0.05%. Since the typical concentrations of SDS used in sample preparation and running buffer for PAGE analysis are 2.4 and 1%, respectively, it means that gel electrophoretic separation of diamond-enriched protein mixtures can be conducted directly without prior removal of the diamond adsorbent, a time-consuming step in sample handling. Apart from SDS, typical MALDI matrixes composed of organic acids such as DHB can also induce complete detachment of the adsorbed proteins from nanodiamonds. Proteome-wide analysis can therefore be performed directly with MADLI-MS, as has been demonstrated in our previous work.25 The observed disruptive effect of SDS on protein adsorption may be rationalized as a result of favorable competition of the organic molecules against proteins for the adsorption sites on nanodiamonds through hydrophobic forces. Alternatively, it may result from the complexation of the detergent molecule with proteins that reduces their adsorbability to diamond. To restore the protein extraction capability, we added methanol to the sample solution to increase the tolerance of the protein adsorption toward SDS. Results of the measurement are shown in Figure 1, where capture of the myoglobin molecules is seen largely reinstated in 40% CH3OH at a SDS concentration up to 0.5%. Since SDS is known to severely deteriorate MS analysis, an important message conveyed by this finding is that direct mass analysis of the

Table 1. Compatibility of Buffer Compositions with Protein Adsorption to Nanodiamonds organic solvents detergents protein dyes alkaline solutions acidic solutions salts others

methanol; acetonitrile 0.01% SDS in DI water; 0.5% SDS in 40% methanol; 5% Triton X-100; 5% CHAPS 0.001% coomasie blue R-250 in DI water; 0.5% coomasie blue R-250 in 40% methanol 1 M NH4HCO3; 0.1% NH4OH 1% formic acid; 1% trifluoroacetic acid 2.5 M NaCl; 1 M Tris-HCl (pH 8) 1.5% glycine; 50% glycerol; 9 M Urea

Figure 3. Protein adsorbability tests of nanodiamonds at pH ∼2 using (a) horse heart cytochrome c and (b) cell lysate. In (a), nanodiamonds with a fixed protein-to-diamond ratio of 1:50 were added to 500 µL of horse heart cytochrome c solutions containing various amounts (0.2, 1, and 5 µg) of proteins and 1% formic acid. Labels at the top of each lane denote control experiments (C) and loading of the diamond fraction (D) after the protein extraction. In (b), 40, 80, and 160 µg of nanodiamonds were added separately to three identical protein solutions containing 1% formic acid and 8 µg of 293T cell lysates. Labels at the top of each lane denote results from direct loading of the cell lysate (Lysate), lysate proteins precipitated by TCA, loading of the diamond (D) and the supernatant (S) fractions after the protein extraction, respectively.

protein-diamond mixtures with MALDI-MS is possible after removal of SDS by several washes of the sample with methanol (Figure S1 in Supporting Information). In the cases that no SDS is present in the sample solution, the affinity of nanodiamonds for the proteins is not adversely affected even in 9 M urea (Figure 2a) and pure acetonitrile (Figure 2b). As some common organic solvents do not corrupt the adsorption capacity, use of these organic modifiers can successfully restore the protein affinity in the presence of high concentrations of adsorption-interfering reagents such as salts and neutral detergents (Table 1). These remarkable properties make the SPEED platform highly compatible with the general practices applied to proteomics research starting with crude protein extracts. We have previously shown that the protein adsorbability of surface-functionalized nanodiamonds peaks at the isoelectric point of each protein in its native form.25 This pH-dependent adsorbability can be exploited favorably in a situation where crude fractionation of a mixture is desired (vide infra). This very

Figure 4. MALDI-FTICR mass spectra of bovine cytochrome c digested with trypsin (a) on diamond and (b) in solution. (*) denotes the peptide masses matched to theoretical trypsinized cytochrome c fragments. Comparison of the sequence coverage of the protein digested on diamond (shadowed region, 77.9% of total) and in solution (underlined region, 89.4% of total) is given in (c).

property, on the other hand, can severely limit the general application of the SPEED platform in many proteomic laboratory practices. Fortunately, nondiscriminative protein adsorption can indeed be achieved in moderately acidic environments with the use of an excess amount (∼50 times total protein weight) of nanodiamonds. Figure 3a shows a result demonstrating the extraction capability of nanodiamonds in a solution containing 1% formic acid (pH ∼2) as a function of protein concentration. As seen, the recovery of the protein (horse heart cytochrome c in this particular case) from these solutions with the SPEED platform is quite satisfactory at all concentrations. The feasibility of this approach is further demonstrated with cell lysates in Figure 3b, where lysate proteins from the 293T human kidney cell line were extracted nonspecifically. In this experiment, aliquots of the cell lysates were first mixed with nanodiamonds of different quantities in 1% formic acid. The mixture was then incubated at room temperature for 5 min, and the resulting solid/liquid phases were separated by centrifugation. As shown in lane 160-S of the figure, almost all proteins in the supernatant are already depleted when the mass ratio of the nanodiamonds to the total proteins was increased to 20:1. Conceivably at pH ∼2, the carboxylate groups on the diamond surface are largely neutralized and most proteins are Analytical Chemistry, Vol. 78, No. 12, June 15, 2006

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Figure 5. MALDI-FTICR mass spectra of on-diamond-digested bovine cytochrome c analyzed (a) without and (b) with an equal volume of 1% formic acid added to the digestion mixture before centrifugation. Both the supernatant (S) and the precipitated nanodiamonds (D) separated by the centrifugation were analyzed. (*) indicates the expected peptide fragments of the trypsinized cytochrome c, and the spectra in each frame are shifted vertically for clarity.

denatured to some extent. A combination of these two effects, interestingly, is sufficient to bring about the precipitation of all protein molecules under this acidic condition. Compared with the TCA precipitation, which typically takes more than 2 h to attain the same result (Figure 3b, lane TCA), the SPEED protocol is significantly faster (just ∼10 min at ambient temperature), less laborious, and also more effective. Site-specific proteolytic digestion prior to MS-based analysis is a standard step as part of a “bottom-up” approach toward protein identification by peptide mass mapping and characterization of posttranslational modifications.1 Such proteolytic digestion can be conducted for proteins bound to nanodiamonds as well. Figure 4 shows a comparison of the results, obtained with MALDI-FTICRMS, for proteolytic digestion conducted on diamond (trace a) and in solution (trace b) for bovine cytochrome c. It is found that the peptide mass spectra of the protein digested with trypsin on nanodiamonds are very similar to those of the same protein digested in solution in terms of sequence coverage. Nearly 80% of the peptides identified in the tryptic digest of the diamondbound proteins matched well with the theoretically predicted fragments (Figure 4c). It is worth noting that, when the tryptic digestion was carried out at pH 9, the majority of the peptide fragments detached spontaneously from the nanodiamond surfaces (trace S in Figure 5a). One may make good use of this feature, which occurs in alkaline environments, for analyzing detached proteolytic peptides 4232 Analytical Chemistry, Vol. 78, No. 12, June 15, 2006

Figure 6. MALDI-FTICR mass spectra of tryptic digests of bovine serum albumin. The digestion was conducted either (a) on diamond or (b) in gel. The sequences of the peptide fragments containing S-pyridylethylated cysteine residue(s) are indicated explicitly for the corresponding peaks.

by on-line LC-ESI-MS after removal of the adsorbents by centrifugation. Alternatively, the detached proteolytic peptide fragments can be readsorbed onto the nanodiamonds with the addition of 1% formic acid that results in nondiscriminative adsorption as described earlier for ensuing MALDI-MS analysis (trace D in Figure 5b). Figure 5 shows this reversible pHdependent adsorbability, a fascinating characteristic of the surfacefunctionalized nanodiamonds in extracting proteins as well as peptides from highly dilute and contaminated solutions. Reductive cleavage of disulfide bonds and ensuing protective alkylation of cysteine residues are routine procedures in peptide mapping of proteins containing inter- or intramolecular disulfide bonds.1 However, the presence of residual reagents from these chemical reactions often hampers proteolytic digestion as well as subsequent MS analysis. In-gel digestion of proteins alleviates this problem by taking the advantage that those chemicals are small molecules, which can be removed easily from the polyacrylamide gel without significant loss of the processed proteins. Such a problem can be similarly (and sometimes more effectively) resolved by on-diamond digestion, in which the proteins attached to nanodiamonds can be washed extensively with DI water to remove residual chemicals prior to enzymatic digestion. Figure 6a shows the result of a test with bovine serum albumin, where the work flow involving reduction, protection, proteolysis, peptide fragment recovery, and final MALDI-FTICR-MS (as detailed in Experimental Section) was conducted without removal of the diamond adsorbents. As seen, the on-diamond digestion result is qualitatively comparable to that of in-gel digestion (Figure 6b). The match of these two results manifests a gel-free approach

Figure 7. (a) SDS-PAGE analysis of urinary proteins extracted with nanodiamonds at three different solution pHs. Results from TCA precipitation for the same sample are shown in the left-most lane for comparison. (b) Sex-dependent protein histogram showing peptide hits of urinary proteins identified in the urine samples from 10 healthy male (blue) and 10 healthy female (red) adults.

toward digestion of proteins containing disulfide bridges using nanodiamonds. The efficacy of the SPEED platform for proteome analysis is finally demonstrated by analyzing human urine, which can be collected noninvasively for clinical diagnosis and biomarker discovery.3,12 Urine contains a variety of contaminants such as urea, uric acids, salts, and other metabolites that interfere strongly with MS analysis of urinary proteins. SPEED is well-suited as a cleanup platform for this purpose. As the protein adsorbability peaks at the isoelectric point of each protein,25 crude prefractionation of the urinary proteins can be established by adjusting the solution pH. Figure 7a shows the SDS-PAGE analysis of urinary proteins and their degradation products extracted at pH 3, 7, and 11. Compared with the total proteins collected by the TCA precipitation, both human serum albumin and the light chain of immunoglobulin (two most abundant proteins in urine) are relatively lower in abundance in both pH 7 and pH 11 fractions. While not all proteins are collected in a favorable fashion, this distinct pH-dependent preference provides a simple way to minimize the interference from these highly abundant proteins in proteome-wide analysis of urine and, possibly, serum as well. Urinary proteins extracted at three different pHs for each sample were identified with a bottom-up LC-ESI-MS/MS approach incorporating the aforementioned on-diamond proteolysis. From 60 independent ESI-MS/MS runs, we identified a total of 125 proteins from 10 female and 10 male urine samples (Table S1 in Supporting Information). On average, 26 proteins were identified in each pH fraction and 51 proteins were identified in three LC injections for a given sample. To assess the relative abundance of the diamond-bound proteins, we counted the number of identified peptides (i.e., peptide hits) for each protein

and found that some components identified as “sex preferential” appear more frequently in the male than the female urine samples or vice versa. Among these proteins shown in Figure 7b, prostatic acid phosphatase, prostaglandin D2 synthase, and prostate-specific antigen are highly expressed in the seminal fluid.29 Therefore, the preferential identification of them in the male urine is very likely a result of contamination from the seminal fluid. However, in accord with previous findings,5 calgranulin B can be identified only in the female urine. The appropriate identification of these sex-related proteins suggests that the SPEED platform in combination with LC-ESI-MS/MS is a promising approach for rapid screening of disease markers from highly complex humoral fluids. CONCLUSION We have developed a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites and have successfully demonstrated its application to mass spectrometric proteome analysis of human urine. The SPEED platform, combining prefractionation, suppression of abundant proteins, preconcentration, purification, and on-particle digestion, streamlines the preparative procedures for targeted MS analysis. More importantly, it integrates seamlessly into SDS-PAGE, MALDI-MS, and LC-ESI-MS, the three indispensable tools in contemporary experimental proteomic research. The proteome analysis of each urine sample can be finished in 8 h. Compared with the conventional approaches such as protein depletion by affinity column, acid precipitation, filtration, dialysis, and 2D SDSPAGE followed by in-gel digestion,5,9-13,30 the SPEED platform is (29) Fung, K. Y.; Glode, L. M.; Green, S.; Duncan, M. W. Prostate 2004, 61, 171-181. (30) Smith, G.; Barratt, D.; Rowlinson, R.; Nickson, J.; Tonge, R. Proteomics 2005, 5, 2315-2318.

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faster and easier to deploy. The platform may find far-reaching applications, not only in proteome analysis of urine but also in search of disease-related biomarkers in other humoral fluids.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. The material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT The research is supported by grants from Academia Sinica and the National Science Council (Grants NSC 92-3112-B-001-012-Y and NSC 94-2113-M-001-049) of Taiwan, Republic of China.

Received for review November 26, 2005. Accepted April 13, 2006.

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