Development of a sample preparation method for fungal proteomics

FEMS Microbiology Letters 247 (2005) 17–22 www.fems-microbiology.org

Development of a sample preparation method for fungal proteomics Motoyuki Shimizu, Hiroyuki Wariishi

*

Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Received 2 October 2004; accepted 14 April 2005 First published online 30 April 2005 Edited by G.M. Gadd

Abstract Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Brown-rot basidiomycetes; Protein extraction; Proteome; Protoplasts

1. Introduction Very recently, the US Department of Energy and Joint Genome Institute completed the whole genomic sequence of the white-rot basidiomycete, Phanerochaete chrysosporium [1] and the data were made available to the public (http://www.jgi.doe.gov/programs/whiterot.htm). Besides this basidiomycetous fungus, whole genomic sequences, or at least draft sequences, for several filamentous fungi have also been completed (http://www.broad.mit.edu/annotation/fungi/fgi/) [2]. Under such research conditions, comparative genomics and proteomics of these fungi might provide greater insight into the cellular system of eukaryotic microorganisms. Genome data are content-independent; however, proteomes are content-dependent and their quality is *

Corresponding author. Tel./fax: +81 92 642 2992. E-mail address: [email protected] (H. Wariishi).

strongly dependent on the extraction efficiency of proteins from the cells. Filamentous fungi are known to possess an exceptionally robust cell wall [3]; thus, cell lysis is the most difficult but crucial step in sample preparation for two-dimensional gel electrophoresis (2-DE). An intracellular survey of iron-regulated proteins for the white-rot basidiomycetes Lentinula edodes and P. chrysosporium was recently reported using a 2-DE technique; however, the number of 2-DE protein spots observed for these fungi were considerably few when compared with those for other organisms [4]. To better determine fungal proteomes, a modified a sample preparation protocol was thought indispensable. Since the cell wall is thought to cause ineffective protein extraction from basidiomycetous cells, the utilization of fungal protoplasts was of great interest. In our previous report, protoplasts of the brown-rot basidiomycete, Tyromyces palustris, were shown to cause metabolic activities similar to those seen with intact mycelial

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2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.021

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cells [5]. Thus in the present study, protoplast cells were prepared from T. palustris mycelial cells and utilized to omit the obstacles caused by cell walls during the extraction of intracellular proteins. Proteomic maps for intracellular proteins from protoplast or mycelial cells were also compared.

2. Materials and methods 2.1. Culture conditions Tyromyces palustris (IFO 30339) was grown from hyphal inocula at 30 C in a stationary culture under air. The MPYC medium used for the present study was supplemented with 1% malt extract, 1% peptone, 0.4% yeast extract, 0.2% casamino acid in deionized water with pH adjusted to 6.0 [6]. After a 3-day incubation, the mycelial cells were collected by filtration and rinsed with deionized water. The cells were used for either a protein extraction or protoplast preparation. 2.2. Protoplast preparation Mycelial cells (1 g, wet weight) were treated with 5 mL of enzyme solution containing 2% (w/v) Novozyme 234, 0.5% (w/v) Zymolyase 20 T, 0.6 M mannitol, and 50 mM maleate (pH 6.0) [6]. After 1 h incubation at 30 C with gentle rocking, the protoplasts were harvested by centrifugation at 750g for 10 min at 4 C. The pellets were then washed by centrifugation with 50 mM potassium phosphate (pH 6.0) containing 0.6 M mannitol (buffer A). Protoplasts were counted using a hemocytometer. Yields of protoplast cells per g (wet weight of mycelia) was (8.0 ± 0.1) · 107. 2.3. Protoplast regeneration The regeneration medium used in this study was MPYC medium containing 0.6 M mannitol and 1.5% low-temperature agarose as previously described [6]. The protoplast solution obtained as described above was diluted to a concentration of 105 cells/mL using buffer A. Aliquots of suspension (100 ll) were added to the regeneration medium and placed on Petri dishes. Protoplasts were incubated at 30 C for 2–3 days and regenerated protoplasts were counted as individual colonies. 2.4. Extraction of intracellular proteins The protoplasts were suspended in SDS buffer containing 4% SDS, 2% DTT, 20% glycerol, 20 mM PMSF, and 100 mM Tris–HCl (pH 7.4). The solution was then heated for 5 min at 80 C and insoluble material was removed by centrifugation (15,000g for 10 min). Four volumes of cold acetone ( 20 C) were added and the

solution was incubated overnight ( 20 C). After centrifugation (15,000g for 10 min), the precipitate was washed with cold acetone ( 20 C) and the pellet was solubilized in urea buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% DTT, 0.5% IPG buffer (pH 3–10 NL; Amersham Bioscience) and a trace of bromophenol blue. The sample was then incubated for 2 h at room temperature and insoluble material was removed by centrifugation (15,000g for 10 min). Samples were split in half; one was used for electrophoresis and the other for measuring protein concentration using a Bio-Rad protein assay kit based on the Lowry method. The extraction of intracellular proteins from mycelial cells was achieved using the methods described above against a mycelial powder prepared from cells frozen under liquid nitrogen, and ground into a fine powder using a mortar and pestle. 2.5. Subcellular fractionation of protoplasts For subcellular fractionation, protoplasts were collected by centrifugation, then burst via an osmotic shock by adding deionized water (0.5 mL). The supernatant and pellet were separated by centrifugation (15,000g), and each fraction was solubilized using SDS buffer. Sample preparation for 2-DE was as described above. 2.6. Two-dimensional gel electrophoresis Isoelectric focusing was carried out with an IPGphor system (Amersham Bioscience). Immobilized pH gradient strips (pH 3–10 NL, 18 cm; Amersham Bioscience) were rehydrated for 12 h, and then 750 lg of protein in urea buffer was focused in four steps at 500 (1 h), 500–1000 (1 h), 1000–8000 (2 h), and 8000 V (8 h). After completion of focusing, strips were equilibrated with buffer containing 6 M urea, 130 mM DTT, 30% glycerol, 2% SDS, and a trace of bromophenol blue then with a buffer containing 6 M urea, 135 mM iodoacetamide, 30% glycerol, 2% SDS, and a trace of bromophenol blue. Strips were loaded onto precast 12.5% homogenous polyacrylamide gels (20 · 20 cm). The lower running buffer contained 385 mM tris, 50 mM glycine, 0.1% SDS, and 0.02% sodium azide, while the upper running buffer was identical except it lacked sodium azide. The system was run at 1000 V and 24 mA per gel. Gel slabs were stained in 7.5% acetic acid solution with 0.0002% SYPRO Red (Takara) and incubated with gentle rocking at room temperature for 1 h. After removal of the staining solution, gels were washed in 7.5% acetic acid solution for 30 min. 2.7. In-gel tryptic digestion In-gel tryptic digestion was performed as previously described [7] with a slight modification. The target spot

M. Shimizu, H. Wariishi / FEMS Microbiology Letters 247 (2005) 17–22

was excised and cut into 2-mm cubes then the gel pieces were transferred into a 1.5-mL microcentrifuge tube and washed with 40% 1-propanol at room temperature for 15 min. After removal of 1-propanol solution, 200 mM ammonium bicarbonate in 50% acetonitrile was added and the sample was incubated at room temperature for 15 min. The gel pieces were then dried and covered with 20 ng/lL modified trypsin (Promega) in a minimal volume of 100 mM ammonium bicarbonate to rehydrate the pieces; they were then incubated at 37 C. After a 12-h incubation, the supernatant was collected, and the gel pieces were extracted once with 100 mM ammonium bicarbonate, followed by two extractions with 80% acetonitrile containing 0.05% trifluoroacetic acid. The supernatant and extracts were combined and concentrated to the required concentrations. 2.8. MALDI-TOF-MS analysis The resulting peptide mixtures were desalted using ZipTips C18 (Millipore) and eluted onto a 96-well MALDI target plate. Two-lL samples on the plate were mixed with 1 lL of 10 mg/mL a-cyano-4-hydroxycinnamic acid solution in 0.1% trifluoroacetic acid in H2 O/acetonitrile (1:1). Samples were then dried at room temperature. Mass spectral data were obtained using a Voyager DE mass spectrometer equipped with a 337nm N2 laser in the positive ion reflectron mode (Applied Biosystems). Spectral data were obtained by averaging 64 spectra, each of which was the composite of 64 laser firings. Internal mass calibration was performed using bradykinin (904.45 Da) and ACTH (2465.75 Da). 2.9. PMF analysis Peptide mass fingerprinting (PMF) was utilized for protein identification by analyzing the sizes of tryptic fragments via the MS-Fit search engine (http://prospector.ucsf.edu) using the entire NCBI protein database. For effective PMF analysis, it was assumed that peptides were monoisotopic and the possibility that methionine residues were oxidized was considered. The fingerprinting method allowed for a maximum of one missed tryptic cleavage per protein. The maximum deviation permitted for matching the peptide mass values was 100 ppm.

the present study contained neither cell wall debris nor spheroplasts (Fig. 1). The average of yield of protoplast per g (wet weight) of T. palustris was (8.0 ± 0.1) · 107 with an average diameter of 4 lm. The regeneration frequency of the protoplasts was 2.23% on MPYC medium containing 0.6 M mannitol and 1.5% low-temperature agarose. 3.2. Comparison of 2-DE patterns between fungal mycelia and protoplasts The amount and quality of proteins extracted from the mycelial cells (5 g wet weight) were affected by the detergent utilized. Total intracellular proteins of 3.8 ± 0.8 mg and 7.2 ± 1.2 mg were obtained when Triton X-114 and CHAPS were utilized, respectively. An identical amount of protein (750 lg) extracted using either Triton X-114 or CHAPS were charged onto 2DE; however, almost no protein spots were observed in the 2-DE gels either with or without the step of acetone precipitation. Since the detergent is known to play a role not only in dissolving proteins but also in cell lysis [9,10], SDS was utilized. SDS is known to be the most powerful detergent for cell lysis [11]. When the protein samples were prepared using SDS, recovery of the total intracellular proteins increased to 34.0 ± 3.2 mg from the mycelial cells (5 g, wet weight). To remove SDS for a smooth 2-DE run, the proteins were precipitated and rinsed with acetone then dissolved in urea buffer [11]. Next, 750 lg of proteins was applied to 2-DE. More protein spots were visualized in the gel compared to other general methods using Triton X-114 and CHAPS (Fig. 2A). The lower recovery of proteins using Triton X-114 and CHAPS might have been caused by the lower effective disruption of the cell wall. SDS extraction coupled with efficient removal of the detergent using acetone provides the best 2-DE map for the basidiomycete T. palustris. However, only 137 protein spots were visualized after SYPRO Red staining, suggesting an inhibitory effect of the fungal cell wall on protein extraction.

3. Results and discussion 3.1. Microscopic observation and regeneration frequency of fungal protoplast Fungal protoplasts extrude through ruptures in the cell wall; thus they completely lack a cell wall [8]. Microscopic observation revealed that protoplasts prepared in

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Fig. 1. Microscopic observation of T. palustris protoplasts.

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Fig. 2. 2-DE profiles of mycelial (A) and protoplast (B) proteins. The same amount (750 lg) of proteins obtained from mycelial and protoplast cells were analyzed using 2-DE, respectively.

To achieve effective cell lysis, we utilized protoplast cells to extract total intracellular proteins. The amount of proteins obtained from (4.00 ± 0.15) · 108 protoplasts, which was the number obtained from 5 g (wet weight) of mycelial cells, was 2.5 ± 0.2 mg. This protein recovery was rather small compared to that from 5 g (wet weight) of mycelial cells using the SDS method (34.0 ± 3.2 mg); however, the regeneration frequency of the protoplasts was only 2.23%. If regeneration was quantitative, 112.1 mg proteins would be expected. Therefore, it was tentatively concluded that the protein recovery from protoplast cells was increased 3-fold compared to that from the mycelial cells using SDS and 16-fold to that from the mycelial cells using CHAPS. Total intracellular proteins from the protoplasts (750 lg) was applied to 2-DE and the proteome map is shown in Fig. 2B. Fig. 2 shows the quantitative comparison of the proteome maps prepared from mycelial and protoplast cells, indicating that protein spots with a wider range of molecular weights and pIs were seen for protoplast sample. Furthermore, no streaking or tailing was observed from the protoplast cells, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress the degradation of proteins during this process. A higher reproducibility of the 2DE map was obtained with protoplast cells, which might be attributed to the constant number of cells. 3.3. Subcellular fractionation using protoplasts Protoplasts were collected by centrifugation then burst through an osmotic shock by adding deionized water. The pellet and supernatant were separated by centrifugation then each fraction was solubilized using SDS buffer. After precipitation by acetone, proteins from either pellet or supernatant fraction were solubilized using the urea buffer for 2-DE.

About 200 protein spots were visualized from the pellet fraction. One protein spot showed the best fit to ATP synthase b-chain via peptide mass fingerprinting (PMF) analysis (Fig. 3A, Table 1). ATP synthase is known to be a mitochondrial membrane protein, suggesting that the pellet fraction contained membrane proteins. Fig. 3B shows the 2-DE map obtained from the supernatant fraction, where more than 300 protein spots were visualized. Comparison of the 2-DE map from the pellet sample with that from the supernatant sample indicated completely different patterns (Fig. 3). The PMF of spot 2 and spot 3 showed the best fit to heat shock protein 70 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, which are known cytosolic soluble proteins (Table 1), suggesting that the supernatant fraction contained soluble proteins. Many membrane-associated proteins are known to possess a high pI [12,13], which is consistent with the data shown in Fig. 3. These results strongly suggest that subcellular fractionation using protoplasts efficiently separates soluble and membrane proteins from the basidiomycetous cells. Since the whole genomic sequence of T. palustris is not known, the PMF method is insufficient for identification of each spot observed on the 2-DE map. Protein identification by determining internal sequences using ESI-MS/MS is now underway. In this study, brown-rot fungus protoplasts were utilized to omit the obstacles caused by cell walls during extraction of intracellular proteins. By using protoplasts, not only effective extraction of intracellular proteins but also simple and efficient subcellular fractionation were achieved. Combined with our recent report showing that protoplasts of T. palustris caused metabolic activities as seen with intact mycelial cells [5], the present data strongly suggest that cellular responses of basidiomycetes could be detected more sensitively using protoplasts because they are single living

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Fig. 3. 2-DE profiles obtained from pellet (A) and supernatant (B) fractions. After protoplasts were burst through an osmotic shock by adding deionized water, the pellet and supernatant were separated by centrifugation. The same amount (750 lg) of proteins obtained from pellet and supernatant fractions were analyzed using 2-DE, respectively. Table 1 Tentative identification of proteins Rank

Protein name

Species

Accession No.a

Spot 1 1 2

ATP synthase b-chain ATP synthase b-chain

Kluyveromyces lactis Saccharomyces cerevisia

P49376 P00830

Spot 2 1 2

Heat shock protein HSS1 Heat shock protein 70

Puccinia graminis Pichia angusta

Spot 3 1

GAPDH

Phanerochaete chrysosporium

a b c d e

MOWSE scoreb

pI c

MWd

Masses matchede

521 499

5.2 5.5

54.1 54.8

5/11 5/11

Q01877 P53623

35800 3630

5.1 5.0

70.6 70.1

8/15 6/15

Q01982

1080

8.1

36.3

4/10

Accesssion number in NCBI database. MOWSE score obtained from MS-Fit (http://prospector.ucsf.edu) search. Theoretical pI. Theoretical mass. Number of peptides matched to the protein in the database with peptides observed by MALDI-TOF-MS analysis.

cells. Although it is necessary to identify proteins produced upon the preparation of protoplasts such as cell wall-generating enzymes and cellular adhesion enzymes, proteomic differential display analysis using protoplast cells might be a useful and sensitive technique for surveying cellular responses. Acknowledgments This research was supported by a Grant-in-Aid for Scientific Research from the Japan Science Promotion Society and Science and Technology Incubation Program in Advanced Regions from the Japan Science and Technology Corporation (to H.W.). References [1] Martinez, D., Larrondo, L.F., Putnam, N., Gelpke, M.D., Huang, K., Chapman, J., Helfenbein, K.G., Ramaiya, P., Detter,

J.C., Larimer, F., Coutinho, P.M., Henrissat, B., Berka, R., Cullen, D. and Rokhsar, D. (2004) Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat. Biotechnol. 22, 695–700. [2] Galagan, J.E., Calvo, S.E., Borkovich, K.A., Selker, E.U., Read, N.D., Jaffe, D., FitzHugh, W., Ma, L.J., Smirnov, S., Purcell, S., Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C.B., Butler, J., Endrizzi, M., Qui, D., Ianakiev, P., Bell-Pedersen, D., Nelson, M.A., Werner-Washburne, M., Selitrennikoff, C.P., Kinsey, J.A., Braun, E.L., Zelter, A., Schulte, U., Kothe, G.O., Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis, M., Mauceli, E., Bielke, C., Rudd, S., Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R.L., Perkins, D.D., Kroken, S., Cogoni, C., Macino, G., Catcheside, D., Li, W., Pratt, R.J., Osmani, S.A., DeSouza, C.P., Glass, L., Orbach, M.J., Berglund, J.A., Voelker, R., Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D.O., Alex, L.A., Mannhaupt, G., Ebbole, D.J., Freitag, M., Paulsen, I., Sachs, M.S., Lander, E.S., Nusbaum, C. and Birren, B. (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868. [3] Ruiz-Herrera, J. (1992) Fungal Cell Wall: Structure, Synthesis, and Assembly. CRC Press, Boca Raton, FL.

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[4] Hernandez-Macedo, M.L., Ferraz, A., Rodriguez, J., Ottoboni, L.M.M. and De Mello, M. (2002) Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: Differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles. Electrophoresis 23, 655–661. [5] Shimizu, M., Kobayashi, Y., Tanaka, H., Wariishi, H., 2005. Transportation mechanism of vanillin uptake through fungal plasma membrane. Appl. Microbiol. Biotech., in press. [6] Eguchi, F., Tashiro, M., Suzuki, T. and Higaki, M. (1990) Preparation and regeneration of protoplasts of edible mushrooms. Mokuzai Gakkaishi 36, 232–240. [7] Rosenfeld, J., Capdevielle, J., Guillemot, J.C. and Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173–179. [8] De Vries, O.M.H. and Wessels, J.G.H. (1972) Release of protoplasts from Schizophyllum com mune by a lytic enzyme

[9]

[10]

[11]

[12]

[13]

preparation from Trichoderma viride. J. Gen. Microbiol. 73, 13– 22. Herbert, B. (1999) Advances in protein solubilization for two-dimensional electrophoresis. Electrophoresis 20, 660– 663. Fountoulakis, M. and Takacs, B. (2001) Effect of strong detergents and chaotropes on the detection of proteins in twodimensional gels. Electrophoresis 22, 1593–1602. Hurkman, W.J. and Tanaka, C.K. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806. Ohlmeier, S., Scharf, C. and Hecker, M. (2000) Alkaline proteins of Bucillus subtilis: first steps towards a twodimensional alkaline master gel. Electrophoresis 21, 3701– 3709. Molloy, M.P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal. Biochem. 280, 1–10.