A Proteomics Strategy To Discover β-Glucosidases from Aspergillus

Nov 20, 2007 - Atlantic Basin Conference Call for Abstracts: Deadline Extended to Oct 2nd. The American Chemical Society has partnered with seven othe...
0 downloads 13 Views 701KB Size
A Proteomics Strategy To Discover β-Glucosidases from Aspergillus fumigatus with Two-Dimensional Page In-Gel Activity Assay and Tandem Mass Spectrometry Kee-Hong Kim,§ Kimberly M. Brown, Paul V. Harris,* James A. Langston, and Joel R. Cherry Novozymes, Inc., 1445 Drew Avenue, Davis California 95618 Received June 8, 2007

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. β-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few β-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover β-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and β-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as β-glucosidases was confirmed by tandem mass spectrometry. Two novel β-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel β-glucosidase (EAL88289) was cloned and heterologously expressed. The expressed β-glucosidase showed far superior heat stability to the previously characterized β-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of β-glucosidases in a high throughput fashion. Keywords: Proteomics tool • in-gel activity • 2-dimensional electrophoresis • β-glucosidases • Aspergillus fumigatus • fungus

Introduction Cellulose is nature’s most abundant polysaccharide, and enzymatic degradation of lignocellulosic biomass into soluble free sugars has drawn considerable attention due to its potential as an alternative energy source after fermentation to ethanol, and as a source of raw materials for the manufacture of high value coproducts as part of an integrated biorefinery.1–3 The cellulose component of lignocellulose can be degraded in vitro by cellulases consisting of three enzyme classes; 1,4-β-Dglucan cellobiohydrolases (cellobiohydrolase, EC 3. 2. 1. 19), 1,4-β-D-glucan 4-glucanohydrolases (endoglucanase, EC 3. 2. 1. 4), and β-D-glucoside glucohydrolase (β-glucosidase, EC 3. 2. 1. 21). Endoglucanases hydrolyze the internal bond of a cellulose polymer and produce ends that are acted upon by a cellobiohydrolase, generating cellobiose. The cellobiose is subsequently degraded by β-glucosidase into glucose.4 Synergistic interaction between cellulase components is well-established, but the mode of synergism in cellulase degradation has focused * To whom correspondence should be addressed. [email protected]. § Current address: Center for Prostate Disease Research, Uniformed Services University of the Health Sciences. E-mail: [email protected]. 10.1021/pr070355i CCC: $37.00

 2007 American Chemical Society

primarily on cellobiohydrolase and endoglucanase.5–12 It has been known for nearly three decades that the addition of β-glucosidase from Aspergillus niger to a cellulase preparation of Trichoderma reesei significantly increases the rate of glucose production by removing inhibitory cellobiose.13 However, the synergistic action of β-glucosidases in cellulose degradation is not well-studied, and only a few β-glucosidases have been characterized despite their importance in promoting the complete degradation of crystalline cellulose to glucose.1,14 Proteomics is an excellent tool in profiling, discovering, and identifying proteins produced in response to a particular cellular environment.15 Traditionally two-dimensional gel electrophoresis (2DGE) has been utilized for proteome profiling,16,17 even though it suffers from certain limitations such as limited dynamic range and inability to efficiently detect certain classes of proteins (e.g., strongly basic). Alternative gel-free methods such as MuDPIT (multidimensional protein identification technology) have been more recently developed as proteomics survey tools, but these also have limitations resulting from extreme sample complexity and the limited scan speeds/duty cycles of the current generation of mass spectrometers.18,19 Journal of Proteome Research 2007, 6, 4749–4757 4749 Published on Web 11/20/2007

research articles Both gel-based and gel-free methods show advantages and disadvantages, but selection of technology depends on the ultimate goal of an analysis. Although 2DGE is known to be a slow process, one can significantly reduce the number of target proteins analyzed by LC-MS/MS if there are simple ways of locating the target enzymes in a 2DGE gel. A typical method using image comparison and selection of target proteins requires multiple 2DGE gels, expensive software, and expertise. Recent studies20–23 have shown the utility of zymography (ingel activity assay) involving protein separation by electrophoresis, in situ assay of enzymatic activities, followed by mass spectrometry for protein identification. After van Tilbeurgh et al.24,25 introduced a soluble substrate analogue (4-methylumbelliferyl β-D-glucoside, MUGlc) for β-glucanases, several attempts were made to detect and isolate cellulase components with in-gel activity assays. However, no one has yet reported a 2DGE in-gel activity assay using complete or near-complete denaturing and renaturing of β-glucosidases from a heterogeneous mixture of proteins. Kyriacou et al.26 performed zymography in isoelectric focusing (IEF) using 4-methylumbelliferyl β-D-cellobioside to detect isoelectric points of cellulase components (CBH, EGI-III) after chromatographic purification. However, they neither employed denaturation methods for protein separation nor renatured cellulase components. More recently, Mathew and Rao27 detected the activity bands of endoglucanase isozymes in nondenaturing PAGE using Congo red as a staining method. The limited availability of genome sequences for filamentous fungi compared to other commonly studied prokaryotes and eukaryotes has been a major difficulty for proteomic analysis of fungi, although the situation has been steadily improving. Aspergillus fumigatus was one of the first filamentous fungi for which a draft genome sequence was available. A. fumigatus produces cellulase components when grown on cellulose28,29 and secretes cellulases including β-glucosidase.30–32 None of these β-glucosidases have previously been cloned. Here, we report the development of a proteomic strategy for detecting in situ β-glucosidase activities in a 2DGE gel from the secreted proteome of A. fumigatus followed by subsequent identification using LC-MS/MS. A gene encoding one of the identified β-glucosidases was cloned and expressed for subsequent characterization.

Materials and Methods Chemicals. All chemicals for protein solubilization, immobilized pH gradient strips, and IPG buffers were from GEHealthcare (Piscataway, NJ). CHAPS was from Pierce Chemical Co. (Rockford, IL). Sypro Orange protein gel stains were obtained from Molecular Probes (Eugene, OR). SDS-PAGE analysis for both 1D- and 2DGE utilized the Criterion electrophoresis cell and Tris-glycine 8–16% SDS-PAGE gels (Bio-Rad, Hercules, CA). Spectroscopic assays were conducted with a spectrophotometer (Molecular Dynamics, Sunnyvale, CA) 96well plate reader. 4-Methylumbelliferyl-β-D-glucoside (MUGlc) and Trinder glucose reagent were obtained from Sigma-Aldrich Co. (Cat. M 9766). Modified porcine trypsin was from Promega. Coomassie Blue R-250, PNGase F, and dithiothreitol were from Bio-Rad. Fungal Strain, Culture, and Fermentation. A. fumigatus was isolated from an environmental sample and its identity verified by sequencing of multiple genomic clones and comparison with the March 2004 draft sequence of A. fumigatus obtained by The Institute for Genomic Research (Rockville, MD). Cultures 4750

Journal of Proteome Research • Vol. 6, No. 12, 2007

Kim et al. were routinely maintained on potato dextrose agar. Inocula for fermentor culture were prepared by inoculating an agar plug (0.5-1.0 cm2) from a plate into 50 mL of medium in a shake flask and incubating for 2 days at 45 °C and 200 rpm. Liquid culture medium contained per liter 20.0 g of glucose, 1.45 g of NH4SO4, 0.42 g of MgSO4 · 7H2O, 0.28 g of CaCl2, 10.0 g of corn steep solids, 2.08 g of KH2PO4, and 0.2 mL of trace metal solution consisting of per liter 10.0 g of citric acid, 58.0 g of ZnSO4 · 7H2O, 10.0 g of CuSO4 · 5H2O, 2.4 g of H3BO3, 27.0 g of MnSO4 · H2O, and 216.0 g of FeCl3 · 6H2O. The pH was adjusted to 5.0 with NaOH. The content of the shake flask was used to inoculate 1500 mL of medium in a stirred reactor vessel. Medium contained per liter 52.0 g of Arbocel B800, 5.0 g of glucose, 3.87 g of NH4SO4, 1.63 g of MgSO4 · 7H2O, 2.08 g of CaCl2, 10.0 g of corn steep solids, 2.80 g of KH2PO4, 1.8 mL of pluronic acid, and 0.75 mL of the same trace metal solution. The reactor was stirred at 1100 rpm with air flow of 1.0 VVM and the pH was maintained at 5.0 by automatic titration with 15% NH4OH or 5 N H3PO4. Fermentation was at 45 °C for 120 h. The culture broth was separated from the biomass by centrifugation at 5000 rpm for 20 min at 4 °C. Protein Preparation. Proteins in the liquid culture medium (500 mL) were concentrated to 5 mL (100 times) and washed by a centrifugal ultrafiltration unit (PM10-Centricon, Millipore) in a low salt buffer (10 mM Tris-HCl, pH 7.4). Proteins were quantified by the micro-BCA protein assay with a microplate reader operating at an absorbance wavelength of 562 nm. Because the secretory proteins from A. fumigatus have N-linked glycosylation on glucanases,33 the proteins were subjected to deglycosylation by N-linked glycosidase (PNGase F). For the N-glycosidase reaction, 100 µg of protein mixture in the low salt buffer was mixed with 5 mU of PNGase F in a 50 µL final reaction volume as per the vendor’s instruction. The mixtures were incubated in a water bath at 37 °C for 16 h. A control sample was prepared in parallel by the same procedures without adding the glycosidase. The deglycosylation reaction was terminated by adding appropriate sample buffers for electrophoresis. Protein samples for 1D- and 2DGE analysis were used from the same batch of glycosidase treatment. Electrophoresis. Proteins (100 µg) were mixed with SDSPAGE sample buffer (62 mM Tris-HCl, pH 6.8, 25% Glycerol, 2% SDS, and 0.01% Bromophenol Blue) for 1D PAGE or isoelectric focusing (IEF) sample buffer (9.5 M urea, 4% (w/v) 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS), 5% (v/v) pH 4–7 and 0.5% (v/v) pH 3–10 Pharmalytes, 5% (v/v) 2-mercaptoethanol, and 0.005% bromophenol blue in double-distilled water) for 2DGE. The resulting mixtures were placed on a rotating wheel overnight at room temperature for extensive solubilization. IPG strips were rehydrated for 16 h at room temperature with IEF sample buffer-protein mixtures in a rehydration tray. IEF was carried out using a Pharmacia Multiphor II apparatus (GE-Healthcare, Piscataway, NJ). IEF with an IPG strip (11-cm, pH 4–7) was performed using a voltage program that first increased linearly from 0 to 500 V for 0.5 h and then increased linearly from 500 to 2000 V over 5.5 h. Voltage was next linearly increased from 2000 to 3500 V over 18 h and then maintained at 3500 V for 24 h for extensive focusing. To avoid recrystallization of urea during the IEF process on IPG strips, the temperature of the cooling bed was maintained at 20 °C. Once IEF was finished, the IPG strips were equilibrated for 10 min in 3 mL of 6.0 M urea, 62.5 mM Tris HCl (pH 6.8), 60 mM iodoacetamide, 2.3% (w/v) sodium dodecylsulfate (SDS), 1% dithiothreitol, 30% (v/v) glycerol, and

In-Gel Activity Assay for β-Glucosidase 0.005% (v/v) bromophenol blue solution. For the seconddimension analysis (SDS-PAGE), each equilibrated strip was placed into a one-well, 11 cm Criterion, 8–16% Tris-HCl gradient gel. SDS-PAGE was carried out in a Multi-Criterion Cell (Bio-Rad, Hercules, CA) with constant voltage (100 V) until the bromophenol blue dye reached the bottom of the gel. Protein Detection. For in-gel activity assay of β-glucosidase, the resulting 2DGE gels were incubated in the SDS-removing buffer (pH 4.0) consisting of 50 mM succinic acid, 50% (v/v) isopropyl alcohol, and 20% (v/v) methanol for 30 min. The gels were washed twice in 50 mM succinic acid buffer (pH 4.0) for 20 min at room temperature followed by 0.5 mM 4-methylumbelliferyl β-D-glucoside (MUGlc) in the 50 mM succinic acid buffer for 20 min. Regions of enzymatic activity were visualized on an UV trans-illuminator. After acquiring an image of the in-gel active zone, the same gel was briefly washed once with distilled water. Then, the same gel was directly stained with fluorescent Sypro Orange for total protein detection and destained using an automated Hoefer Processor Plus staining unit for improved reproducibility and using an adapted protocol.34 Gels were fixed in 40% ethanol, 2% acetic acid, and 0.0005% SDS on a platform rocker for 1 h. Fixing solution was removed and replaced with three repeated wash steps consisting of 2% acetic acid and 0.0005% SDS for 30 min each. Gels were stained for 1.5 h in the dark with 2% acetic acid, 0.0005% SDS, and 0.02% SYPRO Orange stain. Images of the fluorescently stained SDS-PAGE gels were obtained by scanning on a laser scanner imaging system (Storm 860, Molecular Dynamics) using blue fluorescence. Images were viewed and adjusted using ImageQuant version 5.0 (Molecular Dynamics). Gels were further visualized on a Dark Reader blue-light transilluminator with orange filter (Clare Chemical Co, Denver, CO). The 1DGE gels were also stained by Coomassie Blue R-250 after the ingel activity assay. Coomassie-stained SDS-PAGE gels were imaged by scanning with a Scanmaker III (Microtek, Redondo Beach, CA) desktop scanner. Final images were prepared using Adobe Photoshop version 5.5. In-Gel Digestion. The in-gel digestion protocol was adapted from Hellman et al.35 and Rosenfeld et al.36 in order to increase recovery of peptides. All tubes for in-gel digestion process were prewashed with 0.1% TFA in 60% acetonitrile overnight followed by two additional washes with double-distilled water. Visible gel spots from the fluorescent and Coomassie-stained gels were excised for in-gel digestion using a 2 mm biopsy punch (Acu · Punch, Acuderm, Inc., Ft. Lauderdale, FL) and stored at -20 °C in the prewashed tubes until needed. Gel plugs were destained in 100 µL of 50% acetonitrile/50% 100 mM ammonium bicarbonate for 10 min while vortexing and repeated for a total of three washes. One-hundred microliters of acetonitrile was added and removed until the gel plugs were fully dehydrated and had visibly turned white. Acetonitrile solution was removed, and residual solution was evaporated in a ThermoSavant SpeedVac for 5 min. Gel plugs were reduced in 50 µL of 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate for 30 min at 50 °C. After removal of DTT solution, 50 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate was added and incubated for 30 min at room temperature in the dark. Following reduction and alkylation, the gel plugs were washed with 100 µL of 100 mM ammonium bicarbonate for 10 min, dehydrated in 100 µL of acetonitrile, and fully dried under vacuum in a SpeedVac for 30 min. For digestion, 20 ng/mL solution of modified porcine trypsin in 50 mM ammonium bicarbonate was directly added to the dried

research articles gel plugs. Sufficient volume (30–50 µL) of the trypsin digest solution was added to cover the gel plugs and allowed to rehydrate for 5–10 min on ice. If necessary, an additional trypsin solution was added followed by another short incubation to ensure full rehydration, and any excess trypsin solution was removed. After rehydration 5–20 µL of 50 mM ammonium bicarbonate was added, and digestion proceeded overnight at 37 °C. Peptides were extracted from the gel plugs by incubating with 30 µL of 50 mM ammonium bicarbonate, vortexed for 10 min, and placed in a bath sonicator for 5 min. The first extracted peptide solution was removed and saved to a prewashed microcentrifuge tube. A second peptide extraction solution of 5% formic acid in 50% acetonitrile in water was incubated for 10 min, sonicated for 5 min, and briefly centrifuged. This second extraction was added to the recovered peptides of the first extraction and repeated. The final pool of peptides was mixed well and vacuum-evaporated, but not to complete dryness. The final volume of the peptide fragments was adjusted to at least 6 µL with 0.5% formic acid in water. Mass Spectrometry and Protein Identification. A Q-Tof micro, a hybrid orthogonal quadrupole time-of-flight mass spectrometer from Waters Micromass MS Technologies (Milford, MA) was used for LC/MS/MS analysis. The Q-Tof micro was fitted with an Ultimate capillary and nanoflow HPLC system which had been coupled with a FAMOS micro autosampler and a Switchos II column switching device for concentrating and desalting samples (Dionex/LCPackings, Sunnyvale, CA). Five microliters of the peptide fragment mixture from the in-gel digestion was loaded onto a guard column (300 µm i.d. × 5 cm, C Pepmap), fitted in the injection loop, and washed with 0.05% formic acid in water at 40 µL/ min for 2 min using the Switchos II pump. Peptides were separated on a PepMap nanoflow column (75 µm i.d. × 15 cm, C18, 3 µm, 100 Å, Dionex/LC Packings) at a flow rate of 150 nL/min from a split flow of 200 µL/min using the NAN-75 calibrator. A step elution gradient from 5% to 80% acetonitrile in 0.1% formic acid was applied over a 45 min interval. The column eluent was monitored for absorbance at 215 nm and introduced into the Q-Tof micro through an electrospray ion source fitted with a nanospray interface. The Q-Tof micro was fully microprocessor controlled using Masslynx software version 3.2 (Waters Micromass MS Technologies, Milford, MA). Data was acquired in survey scan mode in the mass range of m/z 400-2000. The switching criteria for MS to MS/MS included an ion intensity of greater than 10.0 counts/s and charge states of +2, +3. Analysis spectra of up to 4 coeluting species with a scan time of 1.9 s and interscan time of 0.1 s could be obtained. A cone voltage of 60 V was applied, and the collision energy was programmed to be varied (10–60 V) with respect to the mass and charge state of the eluting peptides. The acquired spectra were combined, smoothed, and centered in an automated fashion, and a peak list was generated. Tandem mass spectra were manually interpreted for de novo sequencing, and the resulting peptide sequences were used to obtain an initial identification using FASTS.37 After the preliminary genome sequence of A. fumigatus became available from The Institute for Genomic Research (Rockville, MD) web site at http:// www.tigr.org, the peak list was used to search against glimmer translated gene models (obtained March 10, 2005) using ProteinLynx Global Server 1.0 (Waters), and Mascot (version 1.8, Matrix Science Ltd.). The gene models were subsequently improved manually and searches performed again. Mass tolerance of precursor and MS/MS for Mascot search was 2.0 and Journal of Proteome Research • Vol. 6, No. 12, 2007 4751

research articles 0.8 Da, respectively. Peptides with Mowse score greater than 41 were accepted as identification. Cloning and Expression of β-Glucosidases from A. fumigatus, Aspergillus oryzae, and A. niger. The gene coding for one of two A. fumigatus β-glucosidases identified in this study (GenPept Accession EAL88289) was cloned, inserted into an expression vector, transformed into Aspergillus oryzae, and expressed as previously described.38 The putatively orthologous β-glucosidase gene from A. oryzae was cloned, expressed and purified as previously described.39 In both cases, the level of β-glucosidase activity in fermentation broths of A. oryzae transformed with an empty expression vector was less than 1% of the β-glucosidase activity of transformants expressing the recombinant protein. The known β-glucosidase from A. niger40 was purified from Novozym 188 (Novozymes). An aliquot was passed through a Q-sepharose Fast Flow column (Amersham Bioscience) using gradient elution (20 mM sodium phosphate, pH 7.0; 0–1 M NaCl). Fractions were pooled based on activity with p-nitrophenyl-β-D-glucopyranoside (pNP-Glc). The pooled fractions were then passed through a second Q-Sepharose Fast Flow column, again using a linear gradient (20 mM sodium phosphate, pH 7.0; 0–0.5 M NaCl). Active fractions were identified as before by activity on pNP-Glc. SDS-PAGE was performed on the active fractions, and showed two primary bands at 120 and 70 kDa. The most active fractions were pooled, and buffer was exchanged into 0.1 M sodium acetate buffer, pH 6.0, with 0.2 M sodium chloride. This solution was then purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 200 gel filtration column (Amersham Bioscience) with isocratic elution (0.1 M sodium acetate buffer, pH 6, with 0.2 M sodium chloride). Fractions were assayed for activity on pNP-Glc. SDS-PAGE was performed on the active fractions, and fractions containing pure β-glucosidase as a single band at 120 kDa were pooled. Assay for β-Glucosidase (Cellobiase) Activity. Cellobiase activity was determined at 65 °C essentially as previously described41 except that the cellobiose concentration was 10 mM and glucose was determined using the Trinder glucose reagent.

Results Observation of in Situ β-Glucosidase Activity. To locate enzymes with β-glucosidase activity, we devised an in-gel activity assay. An in-gel activity assay has two essential prerequisites, the availability of a soluble substrate analogue for the target enzyme (β-glucosidases for this study) and the ability of the enzyme to refold after partial or complete denaturation. The substrate analogue used in this study, MUGlc, contains a fluorescent group for sensitive detection and has small size (338.3 Da) for rapid mass transfer by diffusion into the gel matrix. This analogue for β-glucosidase activity was first introduced by Van Tilbeurgh et al.24 and has been used to study the properties of cellulases by several other groups.25,26,42–44 To identify the β-glucosidase enzymatic activity, the secretome of A. fumigatus was mixed with SDS-PAGE sample buffer containing a reducing reagent (β-mercaptoethanol) and an anionic detergent (SDS). As a positive control, a commercially available source of β-glucosidase from A. niger (Novozym 188) was used to ensure adequate activity for the in-gel activity assay. As shown in Figure 1, multiple β-glucosidase activity bands were observed for A. fumigatus at approximately 120, 95, and 70 kDa. A single β-glucosidase activity band of A. niger was observed at approximately 120 kDa. The same gel was later stained with Coomassie Blue R250 to indirectly measure the 4752

Journal of Proteome Research • Vol. 6, No. 12, 2007

Kim et al.

Figure 1. In-gel detection of β-glucosidase activities following one-dimensional electrophoresis. (A) Activity bands, (B) Coomassie blue stained proteins. Lane 1, 100 µg secreted A. fumigatus proteins; lane 2, 100 µg A. fumigatus secreted proteins after treatment with PNGase F; lane 3, positive control β-glucosidase (300 µg) from A. niger (Novozym 188).

molecular mass of β-glucosidases. Although there was some correlation between the position of activity bands and the position of Coomassie-stained bands, particularly for the 120 kDa region, it was difficult to conclude that a particular stained band correlated with an activity band. On the basis of the banding patterns and intensity of β-glucosidase activity, there was little difference between β-glucosidases with (Figure 1A, lane 2) and without PNGase F treatment (Figure 1A, lane 1). We concluded that β-glucosidase activity could be restored in a gel format after denaturation and renaturation, but onedimensional separation might not provide resolving power for identification by mass spectrometric analysis without further protein purification. In-Gel Activity Assay of β-Glucosidase with 2DGE. For further proteome separation, we employed 2DGE-based proteomic analysis.16 Before proceeding, we examined whether the enzyme activity could be restored after denaturing with urea and the zwitterionic detergent CHAPS as denaturing and solubilizing agents in place of the of the anionic SDS used in the one-dimensional separation (Figure 1). Multiple concentrations of urea (0, 3, 5, 7, and 9.5 M) were tested to find the best condition for restoring β-glucosidase activity (Figure 2). Total elapsed time was tested between 6 and 50 h, and the result for 6 h of running is presented in Figure 2. Although the major band (∼120 kDa) recovered its activity in the presence of up to 9.5 M urea, the intensity of the activity band was decreased by increasing urea concentration. Two other β-glucosidase activity bands (∼95 and 70 kDa) disappeared in the presence of 3 M urea, suggesting that they may be much more sensitive to the denaturing effect of this agent. In 2DGE, two groups of β-glucosidase activity spots (Figure 3A,C,E) were obvious. Those candidates with different masses were designated as BG-I (∼240 kDa) and BG-II (∼120 kDa), respectively, even though we did not know how many different proteins or isoforms might contribute to these activity regions at the moment. Since BG-I was a horizontal smear, the existence of isoforms was unclear. The putative BG-II isoforms were well-separated and formed in spot-train shapes with different pI points, which is a typical format of isoforms with modification in a 2DGE gel. While the location of both β-glucosidase activities (BG-I and -II) remains consistent among 2DGE gels at increasing urea concentration (Figure 3A,C,E), the increasing urea decreased the intensities of enzymatic activity. The profile of secretory proteins was dif-

In-Gel Activity Assay for β-Glucosidase

Figure 2. Tolerance of β-glucosidase to increasing concentrations of urea. Lane 1, 0 M urea; lane 2, 3 M urea; lane 3, 5 M urea; lane 4, 7 M urea; lane 5, 9.5 M urea. Proteins were treated with PNGase F before electrophoresis. Thirty micrograms of proteins was mixed with 5% pH 4–7, 0.5% pH 3–10 Pharmalyte, and SDS sample buffer (lane 1) or 4% CHAPS (lanes 2–5).

ferent in each gel (Figure 3B,D,F), indicating that the urea concentrations determined the level of proteome solubility.16 Since our goal was to locate β-glucosidase activities within the entire secretome, and 7.0 M urea was enough to fulfill our objective, we did not examine the effect of fully denaturing 9.5 M urea in 2DGE proteome separation. Protein Identification. After observation of the β-glucosidase active zones, the same gels were stained by a fluorescent dye to detect other proteins and evaluate the resolution (Figure 3B,D,E). It was clear that the in-gel activity assay was significantly higher in sensitivity to the fluorescent protein staining method (Sypro Orange). Because of sensitivity differences between the two fluorescent detection methods, careful measurement of the exact positions of BG-I and -II spots was performed. By measuring the relative positions of β-glucosidase activity spots based on the distance from the top/bottom and the left/right edges of gel to the center of each active zone, three candidate protein spots were cut from fluorescently stained 2DE gels (BG-I, -II, UK in Figure 3B,D,F) and subjected to in-gel trypsin digestion followed by LC-MS/MS. During the initial method development, the genome sequence of A. fumigatus was not yet completed by the international consortium.45 Hence, we decided to use tandem mass spectrometry for protein identification via de novo peptide sequencing and homology search. A detailed tandem mass spectrum and peptide sequencing are presented in Figure 4. The de novo peptide sequencing was performed as described elsewhere.46,47 The resulting interpreted peptide sequence tags were relatively short, but two sequence tags were obtained from BG-I gel plugs. A homology search with peptide sequences was performed with two independent search engines against the same protein database (Swiss-Prot). First, two sequence tags (HYILNEQEN, ANTIVTIHN) were individually searched using

research articles

Figure 3. Detection of β-glucosidase activities (A, C, E) and total protein (B, D, F) after 2D PAGE of secreted proteins of A. fumigatus grown on cellulose. In-gel activities of β-glucosidases were first observed by hydrolysis of MUGlc followed by Sypro Orange staining. (A and B) Denatured by 3.0 M urea; (C and D) 5.0 M urea, and (E and F) 7.0 M urea. Each gel was loaded with 100 µg of protein. UK represents unknown proteins and BG-I/II represents β-glucosidase as determined by mass spectrometry.

Figure 4. Tandem mass spectrum of the triply charged 496.3 Da (MH+ ) 1486.9 Da) ion derived from BG-I (Figure 3), showing b-ion and y-ion series labeled in the peptide sequence and spectrum. Dashed symbols indicate the fragment b ions either with intensity less than 3-fold than noise signal or without detection. Mass-to-charge ratios of observed b4–7 ions were 414.5, 527.6, 641.6, and 770.6 Da, respectively, and those of measured y3–11 ions were 459.2, 588.2, 716.3, 845.3, 959.3, 1072.4, 1185.5, 1348.5, and 1486.7, respectively. On the basis of MS/MS sequencing, multiple sets of peptide sequences were extracted from the BG-I, BG-II, and UK (Figure 3) derived peptide fragments.

BLAST (NCBI) with an option-“short but nearly exact match”. The best match from the two separate searches agreed and the top hit was β-glucosidase of Aspergillus aculeatus with 100% identity (protein access number P48825).48 An additional search using the FASTS algorithm,37 which fits multiple short query peptides to a single database sequence, gave the same results as the BLAST search (Figure 5). More definitive identifications became possible after the release of the A. fumigatus genome Journal of Proteome Research • Vol. 6, No. 12, 2007 4753

research articles

Kim et al.

Figure 5. Search results of FASTS37,55 with two peptide sequence tags based on tandem mass spectra from BG-I (Figure 3). Identification search was performed with two peptide sequence tags (HYILNEQEN and ANTIVTIHN) against the Swiss-Prot sequence database. (A) List of top scoring hits. The box shows the expectation values. The top scoring hit matches with β-glucosidase (P48825) of A. aculeatus.48 (B) The alignments for the top hit. Two arrows indicate the query sequence tags.

sequence.45 This allowed high-confidence identification of two different glycosyl hydrolase family 3 β-glucosidases (Accession Nos. EAL91070 and EAL88289) and a probable family 27 R-galactosidase from the BG-I spot. Detailed information for the identified peptides is summarized in Table 1. Neither de novo sequencing nor blast matches of the digested peptides from the UK (unknown) spots were confident enough to be accepted as identification. Superior Heat Stability of β-Glucosidase from A. fumigatus. One of the putative β-glucosidases detected by in situ activity staining was cloned from A. fumigatus genomic DNA, inserted into an expression vector, and transformed into Aspergillus oryzae for high-level protein expression. The enzyme was assayed for the ability to degrade cellobiose to glucose at elevated temperature and compared directly with a commercially utilized β-glucosidase from Aspergillus niger (Novozym 188) and another β-glucosidase from A. oryzae. On the basis of sequence comparison, both of these enzymes appear to be orthologues of the A. fumigatus β-glucosidase. The results (Figure 6) demonstrate the vastly superior heat stability of the enzyme from A. fumigatus, which maintains at least 90% of its 4754

Journal of Proteome Research • Vol. 6, No. 12, 2007

original activity for 6 h at 65 °C and 65% of its original activity for 19 h. In contrast, the two other Aspergillus enzymes are completely inactivated within 2–3 h at this temperature.

Discussion In this report, we describe an alternative proteomics strategy to discover β-glucosidases. It takes advantage of the refolding capability of β-glucosidases to allow visualization of their location on a 2DGE gel following in-gel activity assay. By comparing active zone and fluorescently stained 2DGE gel images, a limited numbers of protein candidates were readily isolated and subjected to subsequent mass spectrometric analysis. At least some of the β-glucosidases of the A. fumigatus secretome showed substantial tolerance and refolding capability after complete or near-complete denaturation with reducing reagent, detergents, and urea. To our knowledge, this is the first report of successful in-gel activity assay for β-glucosidase with 2DGE using the typical proteome solubilizing agents. There were several previous attempts to develop an in-gel activity assay for other enzymes with 1D SDS-PAGE,49 nonde-

research articles

In-Gel Activity Assay for β-Glucosidase a

Table 1. Mascot Protein Identifications of BG-I and II Spots peptide sequence

calculated mass, Da

observed mass, Da

BG-I A. fumigatus β-glucosidase (Accession No. EAL91070) SPQLLSVFGYDAK 1423.73 1423.57 GVNVLLGPVVGPLGR 1445.87 1445.71 CANTIVTIHNAGIR 1539.78 1538.60 KGVNVLLGPVVGPLGR 1573.97 1573.77 LNFPGLCVSDAGNGLR 1690.80 1689.63 VDVSAGETTQVQFALNR 1833.92 1833.73 DLSTWDVEAQQWSLQR 1960.93 1960.72 HYILNEQETNRNPGMEDGVEVAAVSSNIDDK 3460.57 3459.12 A. fumigatus β-glucosidase (Accession No. EAL88289) VNDFVNVQR 1090.54 1089.43 EIGAASTVLLK 1100.64 1100.51 GIQDAGVIATAK 1142.63 1142.49 A. fumigatus R-galactosidase (Accession No. EAL85784) YDNCYNEGEEGTPK 1674.65 1674.43

FVAQLTPEEK KFVAQLTPEEK 1288.70 SPQLLSVFGYDAK LNFPGLCVSDAGNGLR a

BG-II A. fumigatus β-glucosidase (Accession No. EAL91070) 1160.61 1160.40 1288.50 71 1423.73 1423.49 1689.81 1689.53

Mowse score

60 51 65 42 76 42 125 57 45 52 52 64

48 76 67

Peptides with Mowse scores greater than 41 are shown. β-glucosidase (Accession No. EAL88289) was cloned in this study.

Figure 6. Hydrolysis of cellobiose at 65 °C by β-glucosidases from A. niger (2), A. oryzae (0), and A. fumigatus (b). The enzyme concentrations in the assay were 15, 29, and 9.2 ng/mL, respectively. Values shown are means of independent duplicate determinations. The maximum standard deviation observed for any measurement was 0.21% glucose conversion. β-Glucosidases from A. oryzae and A. fumigatus are recombinantly expressed in A. oryzae, while β-glucosidase of A. niger is purified from a commercial A. niger fermentation. See Materials and Methods for detailed description.

naturing IEF,25 or PAGE27,50 and 2DGE,51,52 but all those procedures lacked at least one critical chemical for complete denaturation. The use of denaturing chemicals is critical in developing an effective in-gel activity assay for 2DGE because complete or near-complete denaturation of the proteome allows high-resolution proteome separation without prefractionation. Following activity staining and isolation of the target proteins, we have shown that is possible to carry out de novo peptide sequencing of the target proteins by tandem mass spectrometry. This approach using in situ activity staining would be useful for analysis of proteomes for which complete genomic sequences are unavailable.

We also demonstrated vastly superior heat stability of one of the β-glucosidases from the thermotolerant species A. fumigatus compared with closely related β-glucosidases from two mesophilic Aspergillus species, A. niger and A. oryzae. One strategy for increasing the activity of the cellulase systems used for lignocellulose hydrolysis is to increase the reaction temperature and take advantage of elevated reaction rates while simultaneously lowering the probability of microbial contamination. Since cellulases operate in synergy, it is necessary that every component of the cellulase system remains stable at elevated temperature. β-Glucosidases are one of the critical components of a complete cellulase system, and the A. fumigatus β-glucosidase characterized here could readily complement a cellulase system operating above the current limit of about 50 °C for the commonly used commercial cellulases from the fungus T. reesei. It was previously shown that sensitivity to heat- and ureainduced denaturation are closely related.53,54 It may therefore not be a coincidence that one of the β-glucosidases detected here by in situ activity staining following urea exposure is relatively heat stable. If this correlation is generally true, the method we describe might represent a useful system of screening for thermotolerant proteins in cases where a suitable activity staining reagent is available. Abbreviations: LC-MS/MS, liquid chromatography tandem mass spectrometer; MUGlc, 4-methylumbelliferyl β-D-glucoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IEF, isoelectric focusing; 2DGE, two-dimensional polyacrylamide gel electrophoresis.

Acknowledgment. We greatly appreciate the technical assistance of William Albano, David Steurer, David Sternberg, and Sarah Teter. The authors acknowledge Natalie Ahn and Katheryn Resing (University of Colorado) for helpful insights and technical discussions. This research was supported in part by Subcontract No. ZCO-1-30017-02 with The National Renewable Energy Laboratory under Prime Contract No. Journal of Proteome Research • Vol. 6, No. 12, 2007 4755

research articles DE-AC36-99GO10337 with the U.S. Department of Energy. Preliminary sequence data was obtained from The Institute for Genomic Research Web site at http://www.tigr.org. Sequencing of Aspergillus fumigatus was funded by the National Institute of Allergy and Infectious Disease U01 AI 48830 to David Denning and William Nierman, the Wellcome Trust, and Fondo de Investicagiones Sanitarias.

References (1) Duff, S. J. B.; Murray, W. D. Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Bioresour. Technol. 1996, 55 (1), 1–33. (2) Galbe, M.; Zacchi, G. A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 2002, 59 (6), 618–628. (3) Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. The path forward for biofuels and biomaterials. Science 2006, 311 (5760), 484–489. (4) Sternberg, D. β-Glucosidase of Trichoderma reesei: its biosynthesis and role in the saccharification of cellulose. Appl. Environ. Microbiol. 1976, 31, 648–654. (5) Nidetzky, B.; Steiner, W.; Hayn, M.; Claeyssens, M. Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. Biochem. J. 1994, 298, 705–710. (6) Woodward, J. Synergism in cellulase systems. Bioresour. Technol. 1991, 36, 61–75. (7) Eriksson, T.; Karlsson, J.; Tjerneld, F. A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei. Appl. Biochem. Biotechnol. 2002, 101 (1), 41–60. (8) Kim, D. W.; Jeong, Y. K.; Jang, Y. H.; Lee, J. K. Purification and characterization of endoglucanase and exoglucanase components from trichoderma-viride. J. Ferment. Bioeng. 1994, 77 (4), 363– 369. (9) Valjamae, P.; Pettersson, G.; Johansson, G. Mechanism of substrate inhibition in cellulose synergistic degradation. Eur. J. Biochem. 2001, 268, 4520–4526. (10) Medve, J.; Karlsson, J.; Lee, D.; Tjerneld, F. Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes. Biotechnol. Bioeng. 1998, 59, 621– 634. (11) Medve, J.; Stahlberg, J.; Tjerneld, F. Adsorption and synergism of cellobiohydrolase-i and cellobiohydrolase-ii of trichoderma-reesei during hydrolysis of microcrystalline cellulose. Biotechnol. Bioeng. 1994, 44 (9), 1064–1073. (12) Wood, T. M.; McCrae, S. I. The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. Biochem. J. 1978, 171 (1), 61–72. (13) Sternberg, D.; Vijayakumar, P.; Reese, E. T. β-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 1977, 23, 139–147. (14) Kim, E.; Irwin, D. C.; Walker, L. P.; Wilson, D. B. Factorial optimization of a 6-cellulase mixture. Biotechnol. Bioeng. 1998, 58 (5), 494–501. (15) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422 (6928), 198–207. (16) Gorg, A.; Weiss, W.; Dunn, M. J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004, 4 (12), 3665–3685. (17) Fey, S. J.; Nawrocki, A.; Larsen, M. R.; Gorg, A.; Roepstorff, P.; Skews, G. N.; Williams, R.; Larsen, P. M. Proteome analysis of Saccharomyces cerevisiae: a methodological outline. Electrophoresis 1997, 18 (8), 1361–1372. (18) Yates, J. R., 3rd; McCormack, A. L.; Link, A. J.; Schieltz, D.; Eng, J.; Hays, L. Future prospects for the analysis of complex biological systems using micro-column liquid chromatography-electrospray tandem mass spectrometry. Analyst 1996, 121 (7), 65R–76R. (19) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R., III. Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 1997, 69 (4), 767–776. (20) Serrano, S. M.; Shannon, J. D.; Wang, D.; Camargo, A. C.; Fox, J. W. A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics. Proteomics 2005, 5 (2), 501–510.

4756

Journal of Proteome Research • Vol. 6, No. 12, 2007

Kim et al. (21) Lee, K.; Kye, M.; Jang, J. S.; Lee, O. J.; Kim, T.; Lim, D. Proteomic analysis revealed a strong association of a high level of alpha1antitrypsin in gastric juice with gastric cancer. Proteomics 2004, 4 (11), 3343–3352. (22) Choi, N. S.; Yoo, K. H.; Yoon, K. S.; Maeng, P. J.; Kim, S. H. Nanoscale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye. J. Biochem. Mol. Biol. 2004, 37 (3), 298–303. (23) Bok, R. A.; Hansell, E. J.; Nguyen, T. P.; Greenberg, N. M.; McKerrow, J. H.; Shuman, M. A. Patterns of protease production during prostate cancer progression: proteomic evidence for cascades in a transgenic model. Prostate Cancer Prostatic Dis. 2003, 6 (4), 272–280. (24) van Tilbeurgh, H.; Claeyssens, M.; De Bruyne, C. K. FEBS Lett. 1982, 149 (2), 152–156. (25) van Tilbeurgh, H.; Claeyssens, M. Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates. FEBS Lett. 1985, 187 (2), 283–288. (26) Kyriacou, A.; MacKenzie, C. R.; Neufeld, R. J. Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: evidence demonstrating endoglucanase activity by cellobiohydrolase II. Enzyme Microb. Technol. 1987, 9 (1), 25–32. (27) Mathew, R.; Rao, K. Activity staining of endoglucanases in polyacrylamide gels. Anal. Biochem. 1992, 206, 50–52. (28) Loginova, L. G.; Tashpulatov, Z. h. Multicomponent cellulolytic enzymes of thermotolerant and mesophylic fungi related to Aspergillus fumigatus. Mikrobiologiia 1967, 36, 988–992. (29) Trivedi, L. S.; Rao, K. K. Cellulase induction in Aspergillus fumigatus M 216. Indian J. Exp. Biol. 1980, 18, 240–242. (30) Rudick, M. J.; Elbein, A. D. Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second betaglucosidase. J. Bacteriol. 1975, 124, 534–541. (31) Rudick, M. J.; Elbein, A. D. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties of β-glucosidase. J. Biol. Chem. 1973, 248, 6506–6513. (32) Stewart, J. C.; Parry, J. B. Factors influencing the production of cellulase by Aspergillus fumigatus (Fresenius). J. Gen. Microbiol. 1981, 125, 33–39. (33) Elbein, A. D.; Mitchell, M.; Molyneux, R. J. Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus. J. Bacteriol. 1984, 160, 67–75. (34) Malone, J. P.; Radabaugh, M. R.; Leimgruber, R. M.; Gerstenecker, G. S. Practical aspects of fluorescent staining for proteomics applications. Electrophoresis 2001, 22, 919–932. (35) Hellman, U.; Wernstedt, C.; Gonez, J.; Heldin, C. H. Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 1995, 224 (1), 451–455. (36) Rosenfeld, J.; Capdevielle, J.; Guillemot, J. C.; Ferrara, P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 1992, 203 (1), 173–9. (37) Mackey, A. J.; Haystead, T. A. J.; Pearson, W. R. Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences. Mol. Cell. Proteomics 2002, 1 (2), 139–147. (38) Harris, P. V.; Golightly, E. J. Polypeptides having beta-glucosidase activity and polynucleotides encoding same. United States Patent Application 20050214920 A1, 2005. (39) Lamsa, M.; Fidantsef, A.; Gorre-Clancy, B. New variant of a parent beta-glucosidase, having beta-glucosidase activity, useful in degrading or converting cellulose- and hemicellulose-containing biomass. PCT international patent WO20040992280-A2, 2004. (40) Himmel, M. E.; Adney, W. S.; Fox, J. W.; Mitchell, D. J.; Baker, J. O. Isolation and characterization of two forms of beta-D-glucosidase from Aspergillus niger. Appl. Biochem. Biotechnol. 1993, 39–40, 213–25. (41) Langston, J.; Sheehy, N.; Xu, F. Substrate specificity of Aspergillus oryzae family 3 β-glucosidase. Biochim. Biophys. Acta 2006, 1764, 972–978. (42) Graff, M. D.; Van Veen, I. C.; Van der Meulen-Muileman, I. H.; Gerritsen, W. R.; Pinedo, H. M.; Haisma, H. J. Cloning and characterization of human liver cytosolic β-glycosidase. Biochem. J. 2001, 356, 907–910. (43) McKeon, T. A. Activity stain for polygalacturonase. J. Chromatogr., A 1988, 455, 376–381. (44) Saloheimo, M.; Kuja-Panula, J.; Ylo¨ sma¨ ki, E.; Ward, M.; Penttila¨, M. Enzymatic properties and intracellular localization of the novel Trichoderma reesei β-glucosidase BGLII (Cel1A). Appl. Environ. Microbiol. 2002, 68, 4546–4553.

research articles

In-Gel Activity Assay for β-Glucosidase (45) Nierman, W. C.; Pain, A.; Anderson, M. J.; Wortman, J. R.; Kim, H. S.; Arroyo, J.; Berriman, M.; Abe, K.; Archer, D. B.; Bermejo, C.; Bennett, J.; Bowyer, P.; Chen, D.; Collins, M.; Coulsen, R.; Davies, R.; Dyer, P. S.; Farman, M.; Fedorova, N.; Fedorova, N.; Feldblyum, T. V.; Fischer, R., Fosker; N. ; Fraser, A.; Garcia, J. L.; Garcia, M. J.; Goble, A.; Goldman, G. H.; Gomi, K.; Griffith-Jones, S.; Gwilliam, R.; Haas, B.; Haas, H.; Harris, D.; Horiuchi, H.; Huang, J. Q.; Humphray, S.; Jimenez, J.; Keller, N.; Khouri, H.; Kitamoto, K.; Kobayashi, T.; Konzack, S.; Kulkarni, R.; Kumagai, T.; Lafon, A.; Latge, J. P.; Li, W. X.; Lord, A.; Lu, C.; Majoros, W. H.; May, G. S.; Miller, B. L.; Mohamoud, Y.; Molina, M.; Monod, M.; Mouyna, I.; Mulligan, S.; Murphy, L.; O’Neil, S.; Paulsen, I.; Penalva, M. A.; Pertea, M.; Price, C.; Pritchard, B. L.; Quail, M. A.; Rabbinowitsch, E.; Rawlins, N.; Rajandream, M. A.; Reichard, U.; Renauld, H.; Robson, G. D.; de-Cordoba, S. R.; Rodriguez-Pena, J. M.; Ronning, C. M.; Rutter, S.; Salzberg, S. L.; Sanchez, M.; Sanchez-Ferrero, J. C.; Saunders, D.; Seeger, K.; Squares, R.; Squares, S.; Takeuchi, M.; Tekaia, F.; Turner, G.; de-Aldana, C. R. V.; Weidman, J.; White, O.; Woodward, J.; Yu, J. H.; Fraser, C.; Galagan, J. E.; Asai, K.; Machida, M.; Hall, N.; Barrell, B.; Denning, D. W. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 2005, 438, 1151-1156; erratum Nature 2006, 439 (7075), 502. (46) Bernard, K. R.; Jonscher, K. R.; Resing, K. A.; Ahn, N. G. Methods in functional proteomics: two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients, in-gel digestion and identification of proteins by mass spectrometry. Methods Mol. Biol. 2004, 250, 263–282. (47) Lewis, T. S.; Hunt, J. B.; Aveline, L. D.; Jonscher, K. R.; Louie, D. F.; Yeh, J. M.; Nahreini, T. S.; Resing, K. A.; Ahn, N. G. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol. Cell 2000, 6 (6), 1343– 1454.

(48) Kawaguchi, T.; Enoki, T.; Tsurumaki. ; Sumitani, J.-i.; Ueda, M.; Ooi, T.; Arai, M. Cloning and sequencing of the cDNA encoding β-glucosidase 1 from Aspergillus aculeatus. Gene 1996, 173 (2), 287–288. (49) Parry, N. J.; Beever, D. E.; Owen, E.; Vandenberghe, I.; Van Beeumen, J.; Bhat, M. K. Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem. J. 2001, 353, 117–127. (50) Hou, W.-C.; Lin, Y.-H. Activity staining of pectinesterase on polyacrylamide gels after acidic or sodium dodecyl sulfate electrophoresis. Electrophoresis 1998, 19, 692–694. (51) Park, S. G.; Kho, C. W.; Cho, S.; Lee, D. H.; Kim, S. H.; Park, B. C. A functional proteomic analysis of secreted enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography. Proteomics 2002, 2, 206–211. (52) Kaino, S.; Furui, T.; Hatano, S.; Kaino, M.; Okita, K.; Nakamura, K. Two-dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice. Electrophoresis 1998, 19, 782– 787. (53) Feinstein, R. N.; Howard, J. B.; Savol, R. Heat and urea stability of blood catalase of catalase-mutant mouse strains. Experientia 1971, 27 (10), 1152–3115. (54) Pearce, R. J. Heat stability in concentrated and non-concentrated milks–the effect of urea and beta-lactoglobulin levels and the influence of preheating. J. Dairy Res. 1979, 46 (2), 385–386. (55) Graves, P. R.; Kwiek, J. J.; Fadden, P.; Ray, R.; Hardeman, K.; Coley, A. M.; Foley, M.; Haystead, T. A. J. Discovery of novel targets of quinoline drugs in the human purine binding proteome. Mol. Pharmacol. 2002, 62 (6), 1364–1372.

PR070355I

Journal of Proteome Research • Vol. 6, No. 12, 2007 4757