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1 1
2 2 molecule Body
3 sample pre fractionate separation processing analysis informatics Cell culture Validation Organelle fractionation Protein separation Digestion Mass (m/z) LC/mass spec db search
4 : KUpro Database (,, ) () ( ) () (ES, ips, ) ) ( ( () ()
5 nanolc nanolc Ion trap triple Q 4000Qtrap nanolc MALDI spotter MALDI-TOF-TOF nanolc Qstar Qstar Elite nanoesi-qqtof Molecular interaction analysis system Rreal time PCR DNA Micro Array Time laps fluorescent Microscope Laboratory Information Management system DataBASE Data mining soft KeyMolnet MANGO, ipeach Protein Pilot, MRM Maltiquant, Mascot, DeCyder, Gene Springs, etc. 2D-DIGE Laser micro dissection system
6 Auto-2D-Western, a natural PROTEIN CHIP SYSTEM
7 1) 3) 5) 1p/19q LOH- tissues 1p/19q LOH+ tissues 2) Extract Protein & mrna Extract Protein & mrna Quantification using integrated proteomics 2D-DIGE DIGE itraq DNA Micro- array LOH- vs LOH+ tissues 4) Data mining using ipeach/mango ipeach/ MANGO GO analysis KeyMolnet analysis Biological Validation of specific proteins related to chemosensitivity (1) Western Blotting (2)Immuno CC, HC Immnocytochemistry (3) MRM Biological function 6)
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9 62 41 mrna 246 protein Protein and mrna A mrna mrna G
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11 Integrated Proteomics itraq itraq (Raw (Raw data) data) 2D-DIGE (Raw (Raw data) data) DNA DNA microarray (Raw (Raw data) data) Reference databases (RefSeq, Uniprot, Ensenble) Human (Swiss-Prot accesion No.) Human (Enterz gene ID, gene annotations, chromosomal information) 1) Mol. Cell. Proteomics, 2009, 8(10): ( ) 2) Manuscript submitted 2010 ( ) ipeach ipeach 2) 2) (integrated (integrated Protein Expression Protein Analysis Expression Chart) Analysis Chart) MANGO 1) (Molecular Annotation of Gene Ontology) Perl scripts (Convert gene IDs, adding gene annotations and chromosomal information, merge transcriptome and proteome datasets, etc.) Reference databases (RefSeq, Ensenble) Human ( Affymetrix DNA microarray Probe No.) Files for conversion to the human orthologs All taxonomy (Swiss-Prot) All taxonomy (Ensembl) Human (Ensembl) Merge data sets Human (Enterz gene ID, gene annotations, chromosomal information) Human (Swiss-Prot) Integrated data of about 30,000 proteins Java Database Manager (MySQL) GO analysis using GeneSpring GX software Network analysis using Keymolnet application UniProt GOA data set
12 A:tumor B:normal A:B>2 1<A:B<2 A:B<1/2 1/2<A:B<1, Cut off protein protein & mrna mrna
13 ipeach (integrated Protein Expression Analysis Chart) DNAiTRAQ 2D-DIGE Entrez Gene ID LOH-/LOH+ Gene Ontology... 30,000
14 Gene ontology analysis of up-regulated proteins iched Gene Ontology of Up-regulated Proteins in LOH- tissues GO Term p-value Biological process regulation of gene expression 2.57E-08 regulation of transcription 5.36E-07 RNA splicing chromatin assembly cytoskeleton organization and biogenesis negative regulation of histone modification negative regulation of dephosphorylation Molecular function DNA binding 2.86E-07 phosphotransferase activity, phosphate group as acceptor histone binding transcription factor activity drug binding ubiquitin binding transcription regulator activity phosphatase inhibitor activity kinase activity Cellular component spliceosome nuclear speck filopodium centrosome nucleosome protein kinase CK2 complex microtubule organizing center cytoskeleton microtubule cytoskeleton proteasome complex nuclear body
15 (AO) ch1p/19q LOH-/LOH+ DNA LOH-/LOH+ > 1.5 DNA LOH-/LOH+ < 1.5 itraq2d-dige LOH-/LOH+ > 1.2 By KeyMolnet(
16 DNA LOH-/LOH+ > 1.5 DNA LOH-/LOH+ < 1.5 itraq2d-dige LOH-/LOH+ > 1.2 PZK vimentin
17 LOH- AO Vimentin Vimentin Ch1p/19q ProteinX ch1p19q Kinase Protease
18 ipeach ProteinX Vimentin Up-regulated molecular network in chemoresistant Gliomas extracted by ipeach proteinx1 proteinx1 ProteinX proteiny PZK1 PZK Vimentin
19 Vimentin expression in anapalastic oligodendrogeliomas tissues Vimentin LOH- No.1 No.2 No.3 No.4 LOH+ Vimentin No.5 No.6 No.7 A. Immunohistochemistry of AOG tissues AOG tissues were analyzed with vimentin antibody. Nuclei were counterstained with hematoxylin and so appeared blue. LOH- LOH No.8 Normal brain tissue Tubulin B. Western blotting analysis of vimentin in AOG tissues The lysate from AOG tissues were resolved by SDS-PAGE and immunobloted with anti-vimentin antibody.
20 Vimentin & ProteinX expressions in AO/AOA tissues(40samples) and glioma cell lines (1p/19qLOH- vs 1p/19qLOH+) * * AO Vimentin Quantitation of Vimentin in LOH- vs LOH+ tissues U251 LOH- U373 LOH+ A172 U87 * :p<0.05 n=40 LOH+/LOH- Glioma ProteinX Quantitation of ProteinX in LOH- vs LOH+ tissues U251 LOH- U373 LOH+ A172 U87 * :p<0.05 n=40 Actin Western blotting analysis of Vimentin in glioma cell lines Actin Western blotting analysis of ProteinX in glioma cell lines Vimentin ProeinX
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22 RSP ) NEDO 19 (JST) ) JST 1
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