Degree / Specialization:
Ph.D.    1998      Kyoto University / Pharmaceutical Sciences
M.Sc.    1992      Kyoto University / Analytical Chemistry
B.Sc.     1990      Kyoto University / Organic Chemistry

Current Position and Past Employment History:
2010-present  Professor, Grad School of Pharm Sci, Kyoto Univeristy
2010-present  Adjunct Professor, Institute for Advanced Biosciences, Keio University
2006-2010    Associate Professor, Institute for Advanced Biosciences, Keio University  
2006-2010    PRESTO Researcher, Japan Science and Technology Agency (JST)
2001-2003    Visiting Scientist, University of Southern Denmark, Denmark
1992-2006    Eisai Co., Ltd. Japan

Research Summary:
In the last century, Yasushi Ishihama worked on micro- or nano-scale separation biosciences using capillary electrophoresis and chromatography as well as mass spectrometry, and applied them to drug discovery and development in the pharmaceutical industry.

Since 2001, he has focused on proteome analyses based on nanoLC-MS technologies. So far, he and his colleagues have developed several proteomics tools such as nanoLC-ESI needle columns (J. Chromatogr A, 2002), StageTip mini-columns for sample preparation (Nature Protoc., 2007), an emPAI algorithm (Mol Cell Proteomics, 2005) which is currently available as a part of MASCOT, a CDIT approach for absolute quantitation and phosphopeptide enrichment approaches based on IMAC (2005, 2007). They also applied these technologies to Malaria proteome (Nature 2002), chaperonine GroEL interactome (Cell 2005), mouse brain proteome (Nature Biotechnol. 2005) and a target finding study for an antitumor natural small molecule (Nature Chem Biol., 2007).  

After moving to Keio University in 2006, he has focused on two main topics such as phosphoproteome and membrane proteome, and developed hydroxy acid-modified metal oxide chromatography (HAMMOC) for phosphopeptide enrichment and phase transfer surfactant (PTS)- aided protease digestion for membrane proteome, respectively. Based on these techniques, his group carried out several phospho- and/or membrane proteomics projects on human cancer, malaria, plants and bacteria. Recently, his group together with RIKEN Plant Science Center, reported the first large-scale plant phosphoproteome analysis including tyrosine phosphorylation (Mol Syst Biol 2008). He is also interested in computational analysis for large-scale proteomics data in collaboration with Keio SFC bioinformatics group (Mol Cell Proteomics 2009, BMC Bioinformatics 2010 & PLoS Comput Biol 2011).   

In October 2010, he moved to Kyoto University as a full professor of Department of Molecular & Cellular BioAnalysis (MCBA) in Graduate School of Pharmaceutical Sciences, while he still keeps his position in Keio Univ as an adjunct professor of IAB, Tsuruoka. Currently, his group is focusing on technology development for nanoLC-MS systems with meter-long capillary columns as well as phosphoproteomics-based cell signaling network analysis.    

Keywords:  proteomics, nanoLC-MS, mass spectrometry, phosphorylation, membrane proteins.
Web tools:  
Peptide catalog database ‘Pepbase’ (http:// pepbase.iab.keio.ac.jp)
Protein quantitation tool based on emPAI ‘emPAI Calc’ (http://empai.iab.keio.ac.jp)

Selected Publications:

Top cited & the latest publications available from here

1. Yachie N, Saito R, Sugiyama N, Tomita M, Ishihama Y. Integrative Features of the Yeast Phosphoproteome and Protein-Protein Interaction Map, PLoS Comput Biol., 7(1), e1001064, 2011.
2. Use of Stable isotope labeling by amino acids in cell culture (SILAC) as a spike‐in standard in quantitative proteomics, Geiger T, Wisniewski JR, Cox J, Zanivan S, Kruger M, Ishihama Y, Mann M, Nat Protoc, 6, 147–157, 2011.
3. Ujihara S, Oishi T, Mouri R, Tamate R, Konoki K, Matsumori N, Murata M*, Oshima Y, Sugiyama N*, Tomita M, Ishihama Y. Detection of Rap1A as a yessotoxin binding protein from blood cell membranes, Bioorg Med Chem Lett. 20(22), 6443-6, 2010.
4. Kyono Y, Sugiyama N, Tomita M, Ishihama Y*. Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination, Rapid Commun Mass Spectrom, 24(15), 2277-82, 2010.
5. Kyono Y, Sugiyama N, Imami K, Miyazaki S, Ohira M, Tomita M, Ishihama Y*, Development of Titania Particles Used for Phosphopeptide Enrichment in Mass Spectrometry-Based Phosphoproteomics. J. Mass Spectrom. Soc. Jpn. 58(4): 129-138, 2010.
6. Nakagami H, Sugiyama N, Mochida K, Daudi A, Yoshida Y, Toyoda T, Tomita M, Ishihama Y*, Shirasu K*, Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants, Plant Physiol, 153(3), 1161-74, 2010.
7. Imamura H, Yachie N, Saito R*, Ishihama Y, Tomita M, Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data. BMC Bioinformatics., 11(1), 232, 2010.
8. Iwasaki M, Miwa S, Ikegami T, Tomita M, Tanaka N, Ishihama Y*: One-dimensional capillary liquid chromatographic separation coupled with tandem mass spectrometry unveils the Escherichia coli proteome on a microarray scale. Anal Chem., 82(7), 2616-20, 2010.
9. Fujiwara K, Ishihama Y, Nakahigashi K, Soga T, Taguchi H*: A systematic survey of in vivo obligate chaperonin-dependent substrates. EMBO J., 29(9), 1552-64, 2010.
10. Shinoda K, Tomita M., Ishihama Y*: emPAI Calc – for the estimation of protein abundance from large-scale identification data by liquid chromatography-tandem mass spectrometry. Bioinformatics, 26(4), 576-7, 2010.  
11. Imami K, Tomita M, Ishihama Y*: Quantitative Proteome and Phosphoproteome Analyses of Cultured Cells Based on SILAC Labeling without Requirement of Serum Dialysis. Mol BioSyst., 6(3), 594-602, 2010.
12. Imami K*, Tomita M, Ishihama Y: Development of Dual Stable Isotope Labeling by Amino Acids in Cell Culture and Application to Quantitative Proteomics. J. Mass Spectrom. Soc. Jpn. 58(2): 81-87, 2010.
13. Masuda T, Saito N, Tomita M, Ishihama Y*: Unbiased quantitation of Escherichia coli membrane proteome using phase-transfer surfactants. Mol Cell Proteomics., 8(12), 2770-77, 2009.
14. Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T*, Shinozaki K*: Type 2C Protein Phosphatases Directly Regulate AbscisicAcid-Activated Protein Kinases in Arabidopsis. Proc. Nat. Acad. Sci. USA., 106(41): 17588-93, 2009.
15. Masuda T*, Ishihama Y: Sample Preparation Methods for Large-Scale Analysis of Membrane Proteins Using Shotgun Proteomics. J. Mass Spectrom. Soc. Jpn. 57(3): 145-151, 2009.
16. Nock A, Ascano JM, Jones T, Barrero MJ, Sugiyama N, Tomita M, Ishihama Y, Malik S*: Identification of DNA-dependent protein kinase DNA-PK as a cofactor for the forkhead transcription factor FOXA2. J Biol Chem. 284 (30): 19915-26, 2009.
17. Iwasaki M, Takeshi M, Tomita M, Ishihama Y*: Chemical Cleavage-Assisted Tryptic Digestion for Membrane Proteome Analysis. J. Proteome Res. 8(6):3169-75, 2009.
18. Ravichandran A, Sugiyama N, Tomita M, Swarup S*, Ishihama Y*: Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic pseudomonas species. Proteomics, 9(10):2764-75, 2009.
19. Yachie N, Saito R*, Sugahara J, Tomita M, Ishihama Y: In silico analysis of phosphoproteome data suggests a rich-get-richer process of phosphosite accumulation over evolution. Mol. Cell. Proteomics 8(5): 1061-71, 2009.
20. Ishihama, Y*. Development of solid phase extraction mini-columns for proteome analysis. BUNSEKI KAGAKU 57(12): 1011-18, 2008.
21. Sagane K*, Ishihama Y, Sugimoto H: LGI1 and LGI4 bind to ADAM22, ADAM23 and ADAM11. Int J Biol Sci. 4(6):387-96, 2008.
22. Miyamoto K, Hara T, Kobayashi H, Morisaka H, Tokuda D, Horie K, Koduki K, Makino S, Núñez O, Yang C, Kawabe T, Ikegami T, Takubo H, Ishihama Y, Tanaka N*: High-Efficiency Liquid Chromatographic Separation Utilizing Long Monolithic Silica Capillary Columns. Anal Chem. 80(22): 8741-50, 2008.
23. Kyono Y, Sugiyama N, Imami K, Tomita M, Ishihama Y*:  Successive and Selective Release of Phosphorylated Peptides Captured by Hydroxy Acid-Modified Metal Oxide Chromatography. J. Proteome Res. 7 (10): 4585-93, 2008.
24. Shinoda K, Tomita M, Ishihama Y*: Aligning LC Peaks by Converting Gradient Retention Times to Retention Index of Peptides in Proteomic Experiments. Bioinformatics. 24(14):1590-5, 2008.
25. Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K*, Ishihama Y*: Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol. Syst. Biol. 4: 193, 2008.  
26. Imami K*, Ishihama Y, Terabe S: On-line selective enrichment and ion-pair reaction for structural determination of sulfated glycopeptides by capillary electrophoresis–mass spectrometry. J. Chromatogr. A, 1194(2): 237-42, 2008.
27. Ishihama Y*, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D*: Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 9: 102, 2008.
28. Shinoda K, Sugimoto M, Tomita M, Ishihama Y*: Informatics for Peptide Retention Properties in Proteomic LC-MS. Proteomics 8: 787-798, 2008.
29. Masuda T, Tomita M, Ishihama Y*: Phase Transfer Surfactant-aided Trypsin Digestion for Membrane Proteome Analysis. J. Proteome Res. 7 (2): 731–740, 2008.
30. Imami K, Sugiyama N, Kyono Y, Tomita M and Ishihama Y*: Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS using a Calcined Titania/C18 Biphasic Column. Anal. Sci. 24: 161-166, 2008.
31. Ishihama, Y*. Molecular dynamics in cellular signal transduction systems. J. Jpn. Soc. Mechanical Engineers. 111(7) 578-81, 2008.
32. Ishihama, Y*. NanoLC-MS systems in proteomics. Chromatography. 29: 25-31.2008
33. Hashimoto M, Ishihama Y*, Tomita M and Soga T: Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation. Rapid Commun Mass Spectrom 21: 3579-3584, 2007.
34. Monton MR, Tomita M, Soga T and Ishihama Y*: Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings for CE and CE-MS. Anal Chem 79: 7838-44, 2007.
35. Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M and Ishihama Y*: Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol. Cell. Proteomics 6: 1103-9, 2007.
36. Sato T, Ishihama Y and Oda Y*: Quantitative proteomics of mouse brain and specific protein-interaction studies using stable isotope labeling. Methods Mol. Biol. 359: 53-70, 2007.
37. Rappsilber J, Mann M and Ishihama Y*: Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2: 1896-906, 2007.
38. Kotake Y, Sagane K, Owa T, Mimori-Kiyosue Y, Shimizu H, Uesugi M, Ishihama Y, Iwata M and Mizui Y*: Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat. Chem. Biol. 3: 570-5, 2007.
39. Ishihama Y, Wei FY, Aoshima K, Sato T, Kuromitsu J and Oda Y*: Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment. J. Proteome Res. 6: 1139-44, 2007.
40. Ishihama Y*: Optimization of Nano-Liquid Chromatography Coupled with Mass Spectrometry for Proteomics. J. Mass Spectrom. Soc. Jpn. 55: 157-164, 2007.
41. Imami K*, Monton MR, Ishihama Y and Terabe S: Simple on-line sample preconcentration technique for peptides based on dynamic pH junction in capillary electrophoresis-mass spectrometry. J. Chromatogr. A 1148: 250-5, 2007.
42. Wakabayashi Y, Kobayashi M, Akashi-Takamura S, Tanimura N, Konno K, Takahashi K, Ishii T, Mizutani T, Iba H, Kouro T, Takaki S, Takatsu K, Oda Y, Ishihama Y, Saitoh S-i and Miyake K*: A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4. J. Immunol. 177: 1772-1779, 2006.
43. Saito H, Oda Y, Sato T, Kuromitsu J and Ishihama Y*: Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics. J. Proteome Res. 5: 1803-7, 2006.
44. Murata K, Mano N, Asakawa N and Ishihama Y*: Simplified gradient generator for micro- and nano-liquid chromatography. J. Chromatogr. A 1123: 47-52, 2006.
45. Ishihama Y*#, Rappsilber J# and Mann M: Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics. J. Proteome Res. 5: 988-94, 2006.(shared first authors)
46. Kokubu M, Ishihama Y, Sato T, Nagasu T and Oda Y*: Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis. Anal. Chem. 77: 5144-54, 2005.
47. Kerner MJ#, Naylor DJ#, Ishihama Y#, Maier T#, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-Hartl M, Mann M and Hartl FU*: Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122: 209-20, 2005 (# shared first authors).
48. Ishihama Y, Sato T, Tabata T, Miyamoto N, Sagane K, Nagasu T and Oda Y*: Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards. Nat. Biotechnol. 23: 617-21, 2005.
49. Ishihama Y*, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J and Mann M: Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein. Mol. Cell. Proteomics 4: 1265-1272, 2005.
50. Ishihama Y*: Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J. Chromatogr. A 1067: 73-83, 2005.
51. Katayama H, Tabata T, Ishihama Y, Sato T, Oda Y* and Nagasu T: Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics. Rapid Commun. Mass Spectrom. 18: 2388-94, 2004.
52. Rappsilber J#, Ishihama Y# and Mann M*: Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75: 663-70, 2003. (# shared first authors)
53. Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG* and Mann M*: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419: 537-42, 2002.
54. Ishihama Y, Rappsilber J, Andersen JS and Mann M*: Microcolumns with self-assembled particle frits for proteomics. J. Chromatogr. A 979: 233-9, 2002.

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