1. Motoyuki Shimizu, Hiroyuki Wariishi Development of a sample preparation method for fungal proteomics Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Speaker: Yi-Hsuan, Wang Adviser: Ching-Long, Lan Date: Mar. 13, 2012

2. Introduction

3. Whole genomic sequence of the white-rot basidiomycete, Phanerochaete chrysosporium Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78 http://microbewiki.kenyon.edu/index.php/ File:Whiterot.jpg http://microbewiki.kenyon.edu/index.p hp/File:040504062021.jpg

4. Genomic sequence of several filamentous fungi http://www.broadinstitu te.org/scientific- community/science/proj ects/fungal-genome- initiative/fungal-genome- initiative

5. Genome data Content-independent Proteomes Content-dependent extraction efficiency of protein Strongly dependent on the extraction efficiency of protein from the cell

6. Filamentous fungi Possess an exceptionally robust cell wall Cell lysisdifficult but crucial Cell lysis is the most difficult but crucial step in sample preparation for two- dimensional gel electrophoresis (2-DE) Ruiz-Herrera, J. (1992) Fungal Cell Wall: Structure, Synthesis, and Assembly. CRC Press, Boca Raton, FL.

7. An example by using 2-DE technology white-rot basidiomycetes An intracellular survey of iron-regulated proteins for the white-rot basidiomycetes Lentinula edodes P. chrysosporium considerably few The number of 2-DE protein spots were considerably few To modified a sample preparation protocol was thought indispensable. http://en.wikipedia. org/wiki/File:Shiitak egrowing.jpg 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.

8. The modification of a sample preparation idea Cell wall cause ineffective protein extraction The utilization of fungal protoplasts Tyromyces palustris Protoplasts of the brown-rot basidiomycete, Tyromyces palustris, cause metabolic activities similar to intact mycelial cells. http://tupian.hud ong.com/a2_32_7 2_2030000119955 013230072183501 0_jpg.html

9. Aims 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. protoplast mycelial cells Proteomic maps for intracellular proteins from protoplast or mycelial cells were also compared.

10. Materials and Methods

11. 1) Culture conditions 2) Protoplast preparation 3) Protoplast regeneration 4) Extraction of intracellular proteins 5) Subcellular fractionation of protoplasts 6) Two-dimensional gel electrophoresis 7) In-gel tryptic digestion 8) MALDI-TOF-MS analysis 9) PMF analysis

12. 1) Culture conditions Tyromyces palustris (IFO 30339) Contain： MPYC medium 1% peptone1% malt extract 0.4% yeast extract deionized water with pH adjusted to 6.0 30°C a stationary culture under air filtration and rinsed with deionized water a 3-day incubation http://spt.im.ac.cn/ImageStorage/Img--00000958--028.gif

13. 2) Protoplast preparation 2% (w/v) Novozyme 2340.5% (w/v) Zymolyase 20 T 0.6 M mannitol50 mM maleate (pH 6.0) enzyme solution Mycelial cells were treated with 5 mL of enzyme solution Enzyme solution Enzyme solution: 1 h incubation at 30 ° C with gentle rocking Centrifugation at 750g for 10 min at 4 ° C The pellets were then washed by centrifugation Protoplast solution with 50 mM potassium phosphate (pH 6.0) containing 0.6 M mannitol. Protoplast solution Yields of protoplast cells per g was (8.0 ± 0.1) x10 7

14. 3) Protoplast regeneration MPYC medium containing 0.6 M mannitol and 1.5% low-temperature agarose The protoplast solution was diluted to a concentration of 10 5 cells/mL Added to the regeneration medium and placed on Petri dishes Incubated at 30 ° C for 2–3 days

15. SDS buffer The protoplasts were suspended in SDS buffer SDS solution SDS solution: 80°C The solution was heated for 5 min at 80°C Insoluble material was removed by centrifugation cold acetone Add four volumes of cold acetone and incubate overnight CentrifugationPrecipitate was wash with cold acetone urea buffer Pellet was solubilized in urea buffer 2 h Incubated for 2 h at room temperature Samples split in halfElectrophoresis Measuring protein concentration 4) Extraction of intracellular proteins (15,000g for 10 min) (20°C) 4% SDS2% DTTT 20% glycerol20 mM PMSF 100 mM Tris–HCl (pH 7.4) 7 M urea2 M thiourea 4% CHAPS0.5% IPG buffer 2% DTTbromophenol blue (15,000g for 10 min) Bio-Rad protein assay kit

16. The mycelial cells → Mycelial powder Cells frozen under liquid nitrogen Using a mortar and pestle Ground into a fine powder 4) Extraction of intracellular proteins Protoplasts Intracellular proteins from Protoplasts Mycelial cells Intracellular proteins from Mycelial cells Electrophoresis (2-DE) Measuring protein concentration

17. 5) Subcellular fractionation of protoplasts Protoplasts were collected by centrifugation Burst via an osmotic shock by adding deionized water (0.5 mL) The supernatant and pellet were separated by centrifugation (15,000g) Each fraction was solubilized using SDS buffer Sample preparation for 2-DE was done http://brucie.pixnet.net /blog/archives/200806 http://www.clker.com/clipart- eppendorf-tube-with-pellet-1.html

18. pH gradient Immobilized pH gradient strips (pH 3–10 NL, 18 cm) 750 µg 750 µg 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) Strips were equilibrated with buffer: Strips were loaded onto polyacrylamide gels 1000 V and 24 mA The system was run at 1000 V and 24 mA per gel. stained Gel slabs were stained in 7.5% acetic acid solution with 0.0002% SYPRO Red and incubated for 1 h removal of the staining solution wash in 7.5% acetic acid solution for 30 min. 6) Two-dimensional gel electrophoresis 6 M urea130 mM DTT 2% SDS30% glycerol a trace of bromophenol blue 6 M urea135 mM iodoacetamide 2% SDS30% glycerol a trace of bromophenol blue

19. The target spot was excised and cut into 2-mm cubes Transfer into a microcentrifuge tube Wash with 40% 1-propanol at room temperature for 15 min Add 200 mM ammonium bicarbonate in 50% acetonitrile Incubate for 15 min Dry and cover with 20 ng/lL modified trypsin 100 mM ammonium bicarbonate Incubate at 37°C for 12 hours The supernatant was collected The gel pieces were extracted once The supernatant and extracts were combined 7) In-gel tryptic digestion

20. 8) MALDI-TOF-MS analysis The resulting peptide mixtures were desalted and eluted onto a 96-well MALDI target plate 9) PMF analysis Peptide mass fingerprinting (PMF) MS-Fit search engine Entire NCBI protein database http://www.palsystem.com/index.php?id=210 http://nanomed.missouri.edu/institute/facilities/MassSpectrometry.html

21. Result & Discussion

22. 1) Microscopic observation and regeneration frequency of fungal protoplast 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts 3) Subcellular fractionation using protoplasts

23. 1-1) Microscopic observation Fungal protoplasts completely lack a cell wall Protoplasts prepared in the present study contained neither cell wall debris nor spheroplasts Fig. 1. Microscopic observation of T. palustris protoplasts

24. 1-2) Regeneration frequency (8.0 ± 0.1) X 10 7 The average of yield of protoplast per g of T. palustris was (8.0 ± 0.1) X 10 7 with an average diameter of 4 µm 2.23% The regeneration frequency of the protoplasts was 2.23% on MPYC medium containing 0.6 M mannitol and 1.5% low-temperature agarose.

25. 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts 3.8 ± 0.8 mg Triton X-114 : 3.8 ± 0.8 mg 7.2 ± 1.2 mg CHAPS : 7.2 ± 1.2 mg Almost no protein spots Almost no protein spots were observed in the 2-DE gels Protoplasts Intracellular proteins from Protoplasts Mycelial cells Intracellular proteins from Mycelial cells Electrophoresis (2-DE) Measuring protein concentration

26. 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts SDS Detergent : Dissolving proteins & Cell lysis, SDS was utilized. 34.0 ± 3.2 mg Recovery of the total intracellular proteins increased to 34.0 ± 3.2 mg from the mycelial cells More protein spots More protein spots were visualized in the gel

27. 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts The lower recovery of proteins using Triton X- 114 and CHAPS The lower effective disruption of the cell wall SDS extraction + Acetone The best 2-DE map for the basidiomycete T. palustris. 137 protein spots Only 137 protein spots were visualized cell wall An inhibitory effect of the fungal cell wall on protein extraction

28. 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts Mycelial cells ： 5 g of mycelial cells 34.0 ± 3.2 mg (SDS method) (CHAPS : 7.2 ± 1.2 mg) Protoplasts ： ( 4.00 ± 0.15) x 10 8 protoplasts 2.5 ± 0.2 mg the regeneration frequency: 2.23% 112.1 mg proteins 3 -fold 16 -fold

29. 2) Comparison of 2-DE patterns between fungal mycelia and protoplasts 750 µg Wider range of molecular weights and pIs for protoplast sample No streaking or tailing from the protoplast cells 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. Mycelial cells Protoplast cells

30. 3) Subcellular fractionation using protoplasts 2-DE gel 2-DE profiles obtained from pellet (A) and supernatant (B) fractions. PelletSupernatant

31. 3) Subcellular fractionation using protoplasts 2-DE gel 2-DE profiles obtained from pellet fractions. Pellet About 200 protein spots ATP synthase β –chain ATP synthase β –chain A mitochondrial membrane protein Pellet fraction contained membrane proteins Pellet fraction contained membrane proteins NCBI database Mass 2DE Match

32. 3) Subcellular fractionation using protoplasts 2-DE gel 2-DE profiles obtained from supernatant fractions. Supernatant More than 300 protein spots heat shock protein 70 heat shock protein 70 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cytosolic soluble proteins supernatant fraction contained soluble proteins supernatant fraction contained soluble proteins 2 3

33. 3) Subcellular fractionation using protoplasts 2-DE profiles obtained from pellet (A) and supernatant (B) fractions. Pellet : membrane proteins Supernatant : soluble proteins SupernatantPellet

34. Conclusion

35. Brown-rot fungus protoplasts cellwalls Brown-rot fungus protoplasts were utilized to omit the obstacles caused by cell walls during extraction of intracellular proteins Effective extraction of intracellular proteins Effective extraction of intracellular proteins Simple and efficient subcellular fractionation Simple and efficient subcellular fractionation

36. Conclusion Protoplasts of T. palustris caused metabolic activities as seen with intact mycelial cells detected more sensitively Cellular responses of basidiomycetes could be detected more sensitively using protoplasts because they are single living cells.

37. Conclusion using protoplast cells might be a useful and sensitive technique 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.

38. Thank you for your attention