Immuno-Assay (Proteomic and Metabonomic)

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Presentation transcript:

Immuno-Assay (Proteomic and Metabonomic) Global Discovery Proteomics Metabonomics Table 1 Frontal Cortex Hippocampus Frontal Cortex Hippocampus 11 Del vs 10 Control 11 Del vs 10 Control Cohort 1 Cohort 2 Cohort 1 Two-Phase Extraction Two-Phase Extraction 11 Del vs 10 Control 13 Del vs 13 Control 11 Del vs 10 Control Cohort 1 & 2 combined Aqueous Phase Aqueous Phase Total lysis protein extraction Total lysis protein extraction 20 Del vs 21 Control Total lysis protein extraction LC MS/MS LC MS/MS In-gel digestion Acetone precipitation Acetone precipitation Acetone precipitation Immuno-Assay (Proteomic and Metabonomic) Reduction Reduction Reduction Serum Alkylation Alkylation Alkylation Tryptic digestion 11 Del vs 10 Control Tryptic digestion Tryptic digestion Multiplex-Immunoassay Label-free LC MSE Label-free LC MSE Label-free LC MSE Label-free LC MSE PLGS + Elucidator PLGS + Elucidator PLGS + Elucidator PLGS + Elucidator Transcriptomics Frontal Cortex Hippocampus Peptide detected in both replicates and >80 samples Peptide detected in both replicates and >80 samples Peptide detected in both replicates and >80 samples Peptide detected in both replicates and >80 samples Fenelon et al.,2013 Stark et al.,2008 Stark et al.,2008 10 Del vs 10 Control 10 Del vs 10 Control 10 Del vs 10 Control Affymetrix Gene 1.1 ST Array plates Affymetrix GeneChip (MOE430AB – 45K probes) Affymetrix GeneChip (MOE430AB – 45K probes) MSstats MSstats MSstats MSstats Results - Cross Comparison Figure 1A+D 578 proteins: 59 (FDR p*<0.05) 715 proteins: 186 (FDR p*<0.05) 552 proteins: 104 (FDR p*<0.05) 570 proteins 104 (FDR p*<0.05) 52 probe sets significant (FDR, p*<0.05) 2,087 probe sets significant (FDR, p*<0.05) 455 probe sets significant (FDR, p*<0.05) Figure 2: GSEA and Figure 1C: IPA WGCNA: Chromatin assembly, RNA processing, Protein localization, Glycosylation, GO-EA: Oxidative phosphorylation, ATP-synthesis electron transport GO-EA: synaptic transmission, network with DLG4, CAMK2 as node Pathway analysis GO-EA + IPA Pathway analysis GO-EA + IPA Pathway analysis GO-EA + IPA Pathway analysis GO-EA + IPA Targeted Proteomic Validation (SRM) Frontal Cortex and Hippocampus (20 Del vs 20 Control) Table 2 22q11.2 encoded proteins miRNA targets (Global Discovery derived) Differentially regulated proteins (Global discovery derived) Glutamatergic signalling (Transcriptomic HPC network-based) ASD & mental retardation associated proteins Hippocampal network proteins Total lysis protein extraction Acetone precipitation Reduction Alkylation Tryptic digestion Label-free LC MSE Data Analysis Pathway Model Figure 3 Legend: GO-EA = Gene ontology enrichment analysis, WGCNA = weighted-gene coexpression network analysis, IPA = ingenuity pathway analysis