Gold Nanoparticle-Assisted Protein Enrichment and Electroelution for Biological Samples Containing Low Protein ConcentrationsA Prelude of Gel Electrophoresis Anne Wang,‡ Chin-Jen Wu,† and Shu-Hui Chen*,†,‡ Department of Chemistry, and Institute of Nanotechnology and MicroSystem Engineering, National Cheng Kung University, Tainan, Taiwan Received December 26, 2005
Abstract: Protein enrichment is essential for biological samples that contain low protein concentrations, especially for proteomic studies that require sufficient quantities for subsequent MS analysis. Traditional precipitation methods, however, are limited in the sample volume and protein concentration required to cause efficient precipitations. We showed that gold nanoparticles (Au-NPs) can be easily applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins can be directly applied to gel electrophoresis for Au-NP-protein dissociation followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. We compared this method with trichloroacetic acid (TCA) precipitation method, one of the most common precipitation methods, and TCA method showed no enrichment effect for protein samples with large volumes (>2 mL) or with low protein concentrations (4 ppm). Therefore, Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, it is also particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids, under which TCA method is ineffective. Keywords: Au nanoparticle • gel electrophoresis • protein enrichment
Introduction Modern instrumentation and advanced technologies for analyzing proteins have allowed a large scale of proteome to be analyzed in one set of run. The success of the analysis, however, is highly relied on the starting amount of the sample to analyze since the sensitivity of most analytical tools such as 2-D gel and mass spectrometry is still limited. Therefore, sample cleanup and protein enrichment have been a challenging task for proteomic analysis, especially for samples that contain low protein amount. For most samples, protein pre* To whom correspondence should be addressed.
[email protected]. † Department of Chemistry. ‡ Institute of Nanotechnology and MicroSystem Engineering.
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cipitation by organic reagents such as trichloroacetic acid (TCA) is commonly used as the first step to enrich proteins from crude samples since it can tolerate a large amount of complicated biological matrix. TCA precipitation method, however, is limited by a number of factors. For example, it is ineffective when the protein concentration is low or when the sample volume is large. Moreover, TCA precipitates are difficult to dissolve without enzyme digestion when the precipitated amount is large. Enzyme digestion, however, is not desirable when direct protein analysis is needed for the subsequent process. Recent developments in nanotechnology have shown that nanosubjects such as gold nanoparticles (Au-NPs)1-7 and diamond crystals8 can be applied as a sensitive probe for affinity capture of biomolecules. Functionalized Au-NPs are used successfully for the detection of specific DNA1 and protein2-8 molecules and as compoments of biosensors. Recently, bare colloidal Au-NP has been coupled to a mutant cowpea mosaic virus, which contains 60 cysteine residues on the surface.7 While the main goal of this study was to use the virus capsid as a template to create a network to design nanodevices, a purification process by gel electrophoresis was developed to separate Au-NP-containing viral nanoblocks from the free Au-NP. In their method, thioctic acid was added to the mixture prior to gel loading to prevent Au-NP precipitation in the well and to assist the mobility of both the complex and the free Au-NP.7 Instead of purifying Au-NP-modified complex, we are aiming at using Au-NP as a prelude of gel electrophoresis to concentrate proteins from a large volume of dilute biological fluid such as urine. Unlike culture cells or other biological fluids, the amount of proteins present in urine is generally low but urine samples have attracted great proteomic research interests because it may contain marker proteins released from kidney or other organs. Despite of low protein concentration, a large volume of urine sample can be collected without intrusive sampling. We showed that Au-NP can be used to aggregate a broad range of proteins in addition to those containing a high percentage of cysteine residues. By directly applying the dissolved precipitates to the gel well, Au-NP-aggregated proteins can be dissociated from nanoparticles and the free proteins can be separated by gel electrophoresis. We also investigated the Au-NP-assisted precipitation with and without reduction/alkylation and the efficiency of the proposed method was compared with trichloroacetic acid (TCA) precipitation method. 10.1021/pr0504844 CCC: $33.50
2006 American Chemical Society
technical notes Experimental Section Chemicals and Reagents. Sodium citrate, bovine serum albumin (BSA), β-casein and D,L-Dithiothreitol (DTT) was obtained from Sigma (St, Louis, MO). HAuCl4 was from Aldrich (St, Louis, MO); iodoacetamide was purchased from Fluka (Buchs, Switzerland). All reagents were of the highest grade available. The CE water was deionized distilled water filtered through a Barnstead E-pure system, and had a resistance over 18.0 MΩ/cm. Urine samples collected from clinical patients were obtained from National Cheng Kung University Hospital and the total protein concentration was determined to be around 100 ppm by Lowry assay. Synthesis and Characterization of Gold nanoparticles. All glass wares used for the preparation of colloids were thoroughly washed with aqua regia (3:1 HNO3-HCl), rinsed extensively with distilled water, and then dried in an oven at 70 °C for 2 h. Gold colloids were prepared by sodium citrate reduction of HAuCl4‚3H2O as reported earlier.9 Briefly, a volume of 100-mL sample of 1 mM HAuCl4 was brought to a vigorous boil with stirring in a round-bottom flask fitted with a reflux condenser and 10 mL of 38.8 mM sodium citrate (pH 6.6) was rapidly added to the solution. The solution was boiled for another 15 min, during which the color of the solution was changed from pale yellow to brightly red. The solution was allowed to cool to room temperature with continued stirring and then, the product was stored at 4 °C until further use. According to the literature,10 the Au-NP solution prepared in this manner was estimated to have a concentration around 11.6 nM. The diameter of the colloids was determined by transmission electron microscopy (TEM). A small drop of the colloidal gold was placed on a grid, and excess solution was wicked away by a filter paper. The grid was subsequently dried in air and imaged on a JEOL JEM 1200-EX transmission electron microscope and the accelerating voltage was 80 keV. Protein Concentration. β-casein and BSA were prepared in DI water at a final concentration of 50 ppm. If reduction/ alkylation was to be performed, then the solution (100 µL) was added with 1M DTT (1 µL), heated at 95 °C for 5 min to reduce the disulfide bond, and then the resulting cysteine residues were alkylated with 500 mM iodoacetamide in the dark for 30 min. For TCA precipitation, reduction/alkylation must be performed before the enrichment. Unless specified, the buffer containing 50% TCA was added into the protein solution to yield a final TCA percentage of 20% and the solution was subsequently centrifuged at 15 300 × g for 10 min. After removing the supernatant, the precipitate was washed with 150 µL of 10% TCA once and then 200 µL of DI water twice and finally dissolved in 20 µL of DI water. For Au-NP enrichment, an aliquot of Au-NP was added into the protein solution and the solution was placed in a refrigerator at 4 °C until the precipitate was formed. Subsequently, the mixture was centrifuged and the precipitate was washed with 200 µL of DI water three times and finally dissolved in 20 µL of DI water by brief sonication. For urine samples, the same procedures for both TCA and Au-NP precipitation were followed and the amount of reagents required was calculated based on the total protein concentration in urine determined by Lowry assay. One-Dimensional Gel Electrophoresis. A volume of 20 µL of the prepared protein standard, urine sample, or protein marker was added with 4 µL of gel loading buffer composed of 10% SDS, 350 mM Tris, 30% glycerol, 0.175 mM bromophenol and 6% 2-mercaptoethanol, and then the mixture was loaded
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into the gel composed of 10-15% sodium dodecyl sulfate (SDS)-polyacrylamide. Gel separation was proceeded under a voltage of 90-120 V at 4°C for 4-6 h. After 1D electrophoresis, protein bands were stained by Coomassie Brilliant dyes. In-Gel Digestion and MS Detection. Protein spots were excised from 1D SDS-PAGE gel, destained twice with 1 mL of 100 mM ammonium bicarbonate buffer containing 40% acetonitrile (ACN) for 15 min. The destained gel pieces were then dried under vacuum, reduced with 50-100 µL of 10 mM DTT prepared in 100 mM ammonium bicarbonate at 57 °C for 1 h, and then followed by alkylation with 50-100 µL of 50 mM iodoacetacid prepared in 100 mM ammonium bicarbonate at room temperature for 30 min in the dark. The reduced samples were washed with 100 mM ammonium bicarbonate containing 40% ACN once and DI water twice to remove excess reagents and then dried under vacuum. A volume of 2-10 µL of 0.1 mg/ mL trypsin solution (Promega) dissolved in 100 mM ammonium bicarbonate was added to each sample, and the solution was incubated at 37 °C for overnight. After digestion, the tryptic peptides were extracted with 50-100 µL of 50% ACN three times. The tryptic peptides were analyzed by a Q-TOF mass spectrometry (Q-TOFmicro, Micromass) equipped with a nanoflow HPLC system (LC Packings, Amsterdam, Netherlands). Briefly, a tryptic digest solution (1 µL) was injected onto a column (NAN75-15-03-C18-PM; 75 µm id and 15 cm in length) packed with C18 beads (3 µm, 100-Å pore size, PepMap). Mobile-phase buffer A consisted of 0.1% formic acid and 5% acetonitrile; mobile-phase buffer B consisted of 95% acetonitrile and 0.1% formic acid. The peptides were separated using a linear gradient of 0-70% solvent B over 40 min at a flow rate of 200 nL/min. For sequencing, the MS/MS spectra were obtained by a survey scan and automated data-dependent MS analysis was carried out using the dynamic exclusion feature built into the MS acquisition software. Each MS scan was followed by four MS/MS scans of the first-four most intense peptide mass peaks to obtain as many CID spectra as possible; the peptide sequences were identified using Mascot Search (www.matrixscience.com). The search results that were within the list of significant hit were regarded as identified proteins, and all results were further verified by manual interpretation.
Results and Discussion The molar ratio for Au-NP-protein aggregation was first optimized. Through a systematic study using 5:1, 10:1, 50:1, and 100:1 molar ratios of protein/Au-NP, the capture efficiency was found to reach a plateau as the molar ratio is beyond 10:1 protein/Au-NP. Therefore, all experiments shown in this study were performed at 50:1 molar ratio of protein/Au-NP without further optimization. Au-NP Precipitation of Standard Proteins. Standard proteins were used to demonstrate the applicability of Au-NPassisted protein enrichment and electroelution by gel electrophoresis. To compare the Au-NP enrichment effect with TCA method, both precipitation methods were performed side by side using 100 µL of 50 ppm BSA or β-casein, which was equivalent to 5 µg for each protein. For TCA precipitation, there was just very few white precipitates adsorbed on the wall surface and we had tried to recover these precipitated proteins on the wall by carefully dissolving them in 20 µL DI water. Normally, an amount beyond 20-30 µg of total proteins is required to form obvious TCA precipitates. Unlike TCA precipitation, Au-NP precipitates were clearly formed with even Journal of Proteome Research • Vol. 5, No. 6, 2006 1489
Protein Enrichment
technical notes
Figure 2. Gel electrophoresis of β-casein. Lane 1 was loaded with the protein marker; Lane 2 was loaded with 20 µL of the protein standard (50 ppm) without enrichment; Lane 3 was loaded with 20 µL solution enriched from 100 µL protein standard by 20% TCA; Lane 4 was loaded with 20 µL solution enriched from 2 mL protein standard by 10% TCA; Lane 5 was loaded with 20 µL solution enriched from 2 mL protein standard by 20% TCA; Lane 6 was loaded with 20 µL solution enriched from 2 mL protein standard by 30% TCA.
Figure 1. Gel electrophoresis of (A) β-casein and (B) BSA. Lane 1 was loaded with the protein marker; Lane 2 was loaded with 20 µL of the protein standard (50 ppm) without enrichment; Lane 3 was loaded with 20 µL solution enriched from 100 µL of the protein standard by TCA method; Lane 4 was loaded with 20 µL solution enriched from 100 µL of the protein standard by Au-NP with prior reduction/alkylation; Lane 5 was loaded with 20 µL solution enriched from 100 µL of the protein standard by Au-NP without prior reduction/alkylation. The ratios used for the precipitation were 20% TCA and 50:1 molar ratio of protein/ Au-NP.
just 5 µg of total protein and the precipitate could be easily dissolved in 20 µL DI water by brief sonication. For comparison, 20 µL of un-enriched standard solution was also loaded into the gel. As shown in Figure 1A, β-casein (25 KD) band was clearly observed at the same migration distance for all unenriched, TCA precipitated, and Au-NP-precipitated samples, indicating that the protein band on the gel was for free proteins. In addition to the migration distance, a blue color remained in the gel well after electro-elution, indicating that Au-NPaggregated proteins could be dissociated from nanoparticles by dissolution and electroelution, and therefore, only free AuNP were retained in the gel well. For BSA (66 KD) that contains 35 cysteine residues, as shown in Figure 1B, a shift in the migration distance for both TCA precipitated and Au-NPprecipitated proteins was noticed due to protein denaturing by reduction/alkylation. Without reduction/alkylation performed before Au-NP precipitation, BSA protein was still detected with no shift in migration distance, indicating that both denatured and un-denatured proteins could be captured and released by Au-NP enrichment method. On the basis of the band intensity shown in Figure 1B, however, the band deduced from the un-denatured BSA was weaker than that deduced from denatured BSA, and this could be due to incomplete release of proteins from Au-NP aggregation. Compared to the band without any enrichment shown in Figure 1A and B, the band intensity deduced after either TCA or AuNP enrichment was enhanced due to the enrichment. Moreover, it is noticeable that the factor of enrichment is indifferentiable between TCA and Au-NP aggregation methods for 100 µL of protein solution. 1490
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Figure 3. Gel electrophoresis of urine proteins. Lane 1 was loaded with the protein marker; Lane 2 was loaded with 20 µL of the urine without enrichment; Lane 3 was loaded with 20 µL solution enriched from 200 µL urine by TCA method; Lane 4 was loaded with 20 µL solution enriched from 5 mL-diluted urine by TCA method; Lane 5 was loaded with 20 µL solution enriched from 10 mL-diluted urine by TCA method; Lane 6 was loaded with 20 µL solution enriched from 10 mL-diluted urine by Au-NP without reduction/alkylation; Lane 7 was loaded with 20 µL solution enriched from 15 mL-diluted urine by Au-NP without reduction/alkylation; Lane 8 was loaded with 20 µL solution enriched from 15 mL-diluted urine by Au-NP with reduction/ alkylation. The ratios used for the precipitation were 20% TCA and 50:1 molar ratio of protein/Au-NP.
We had tried to enrich from a larger sample volume to gain a greater amount of proteins for analysis but TCA method failed to cause aggregation. As shown in Figure 2, in which all enrichments were performed from 50 ppm β-casein standard, the band intensity was enhanced after 20% TCA enrichment from 100 µL of the standard sample compared to that deduced from 20 µL standard sample directly loaded into the gel without enrichment. When the sample volume for enrichment was increased to 2 mL, however, the band intensity obtained became even weaker than that obtained from 20 µL standard without enrichment, indicating that no enrichment effect was gained from 2 mL standard solution. We had tried different TCA percentages for precipitating 2 mL of protein solution. As shown in Figure 2, however, the band intensity did not differ significantly for 10, 20, and 30% of TCA. These results clearly indicate that TCA precipitation method is ineffective for a large volume of biological fluids. TCA method was not only ineffective for large volumes but also for low protein concentrations. We had investigated TCA precipitation from 200 µL and 1 mL solution both containing 4 ppm of proteins and no tiny precipitates adsorbed on the wall could be observed for either
technical notes
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Table 1. Proteins Identified from the Gel Bands Indicated in Figure 3 band no.
1
protein name
alpha-1-antitrypsin precursor gi|177836) Ig gamma-2 chain C region (gi|121043) unnamed protein product (gi|28590)
2
Human Serum Albumin gi|4389275)
3
protein Len,Bence-Jones (gi|229528)
immunoglobulin kappa light chain (gi|3169770) anitubulin IgG1 kappa VL chain (gi|1911815) Proapolipoprotein (gi|178775)
case. For Au-NP, the enrichment is still very promising at low concentrations and large volumes and will be further demonstrated in urine samples. Au-NP Precipitation of Urine Proteins. The use of Au-NP for protein concentration was further applied for the enrichment of urine proteins. To investigate the usefulness of Au-NP enrichment for biological fluids that contain extremely low protein concentrations, 200 µL of the urine sample (100 ppm) was diluted to final volumes at 5, 10, and 15 mL, which correspond to 4, 2, and 1.33 ppm of total protein concentration, respectively. The undiluted and diluted urine samples were enriched by Au-NP as well as by TCA method. As shown in Figure 3, with TCA method, the enrichment effect was only gained from 200 µL urine; but no enrichment effect could be gained from either 5 or 10 mL of diluted urine samples. With Au-NP enrichment, on the other hand, the protein could be enriched completely from either 10 mL or 15 mL of diluted urine samples. The total protein concentration in 15 mL of diluted urine is as low as 1.33 ppm and this observation indicates that Au-NP aggregation is not affected by low protein concentrations and large sample volumes. Although the urine volume investigated was only up to 15 mL, we believe that AuNP aggregation can be applied to even greater volumes of biological fluids with more diluted protein concentrations since there was no sign of limitation up to the stage of experiment performed. We had also compared the Au-NP enrichment with and without reduction/alkylation performed prior to the enrichment. As shown in Figure 3, most of the band profile deduced with and without reduction/alkylation was similar. It is noticeable; however, that several bands such as band 2 were detected only with reduction/alkylation reaction performed before the enrichment (Lanes 3 and 8). Therefore, band 2 was expected to contain proteins with a high percentage of cysteine residues
no. of cysteine
peptide sequence
0 0 0 0
LSSWVLLMK ITPNLAEFAFSLYR ELDRDTVFALVNYIFFK VVSVLTVVHQDWLNGK
1 0 1 0 0 0 1 0 1 0 1 2 0 0 1 0 1 0 0 0 0 0 0
QNCELFEQLGEYK HPYFYAPELLFFAK ALVLIAFAQYLQQCPFEDHVK LVNEVTEFAK HPDYSVVLLLR VPQVSTPTLVEVSR DVFLGMFLYEYARQNCELFEQLGEYK HPYFYAPELLFFAK RPCFSALEVDETYVPK VFDEFKPLVEEPQNLIK ALVLIAFAQYLQQCPFEDHVK MPCAEDYLSVVLNQLCVLHEK FQNALLVR PADLPSLAADFVESK QNCELFKQLGEYK DSTYSLSSTLTLSK VYACQVTHEGLSSPVTK TVAAPSVFIFPPSNQQLK. TVAAPSVFIFPPSDEQLK TVAAPSVFIFPPSNEQLK DSTYSLSSTLTPSK FSGSGSGTDFTLKISR SGTASVVNLLNNFYPR
0 0
QGLLPVLESFK VSFLSALEEYTK
in their sequence so that the migration distance was shifted after protein denaturing. The protein identity can be deduced from MS analysis of the in-gel digested proteins. Without any trouble, we had been able to perform in-gel digestion and MS analysis with three protein bands indicated in Figure 3 and a total of 8 proteins were identified as tabulated in Table 1. Human serum albumin is the only protein deduced from Band 2 and as expected, it contains as many as 35 cysteine amino acid residues and six of them are covered in the identified sequences. The rest of six proteins that were identified from either band 1 or band 3 contain no more than 10 cysteine amino acid residues and three of them are covered in the identified sequences. Generally speaking, these results indicate that Au-NP enrichment is generally applicable to a broad range of proteins and performing Au-NP enrichment with and without reduction/alkylation can further assist the identification of proteins that contain high percentage of cysteine residues. Spectroscopic Investigation of Au-NP Aggregates. Using TEM and UV absorbance spectroscopy, we had also examined the microenvironment of Au-NP -aggregated protein solution. As shown in Figure 4, the UV spectroscopy shows that the absorbance maximum of Au-NP was shifted from 520 to 527 nm after adding protein molecules, which indicates the formation of Au nanoparticle aggregates. TEM shown in Figure 5 further reveals that the diameter of free Au-NP was around 13 nm and the diameter of Au-NP aggregates was around 100 nm, which is equivalent to 1000 Au-NP in the aggregate. The decreased in absorbance as protein was added is due to the dilution of the solution from 720 µL to 1 mL when making the measurement. There was color change from bright red to purple when the aggregation was formed. These Au-NP/protein aggregates act as a nucleation core of the precipitate. It is wellknown that the surfactant molecule such as citrate ions plays Journal of Proteome Research • Vol. 5, No. 6, 2006 1491
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Protein Enrichment
Figure 4. UV absorbance spectra of (A) free Au-NP solution and (B) Au-NP solution containing 50:1 molar ratio of casein-Au-NP.
and many other advantages demonstrated here, Au-NP-assisted protein enrichment can be considered as a feasible alternative step in sample preparation to couple with various subsequent protein treatments, including but not limited to gel electrophoresis. We believe that there are tremendous opportunities to be explored in applying nano techniques for sample preparation in proteomics, one of challenging tasks in this technology-driven science.
Figure 5. TEM photographs of (A) free Au nanoparticles and (B) Au nanoparticle solution containing 50:1 molar ratio of urine protein/Au-NP.
important roles in keeping Au-NP suspended in the buffer solution and this implies that the protein molecule may either replace the surfactant molecule or interact with the adsorbed surfactant molecule to induce further conjugations and aggregations. It is interesting to note that the formed aggregates are easy to redissolve in a buffer solution for subsequent analysis and this could be due to a higher ionic strength involved in the aggregation. On the contrary, TCA precipitation involves more hydrophobic interactions and therefore it is difficult to dissolve when the amount precipitated is large.
Conclusion We showed that Au-NP can be easily used to enrich proteins from a large volume of urines that contain low protein concentrations, under which traditional TCA method failed to cause precipitations. In view of the wide availability of Au-NP
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Acknowledgment. The authors would like to thank Dr. Nan-Haw Chow in Pathology Department of National Cheng Kung University Hospital for collecting patient urines. This work was supported by National Science Council in Taiwan. References (1) Zeng, M.; Huang, X. J. Am. Chem. Soc. 2004, 126, 12047-12054. (2) Teng, C.-H.; Ho, K.-C.; Lin, Y.-S.; Chen, Y.-C. Anal. Chem. 2004, 76, 4337-4342. (3) Zhang, C.-X.; Zhang, Y.; Wang, X.; Tang, Z.-M.; Lu, Z.-H. Anal. Biochem. 2003, 320, 136-140. (4) Thanh, N. T. K.; Rosenzweig, Z. Anal. Chem. 2002, 74, 16241628. (5) Yin, K.-B.; Qi, B.; Sun, X.; Yang, X.; Wang, E. Anal. Chem. 2005, 77, 1624-1628. (6) Zhang, C.; Zhang, Z.; Yu, B.; Shi, J.; Zhang, X. Anal. Chem. 2002, 74, 96-99. (7) 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. (8) Soto, C. M.; Blum, A. S.; Wilson, C. D.; Lazorcik, J.; Kim, M.; Gnade, B.; Ratna, B.-R. Electrophoresis 2004, 25, 2901-1906. (9) Li, Y.-T.; Liu, H.-S.; Lin, H.-P.; Chen, S.-H. Electrophoresis 2005, 26, 4743-4750. (10) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509.
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