Chromosome 5. Kyoto HUPO Initiative Assembly. Peter Horvatovich 1, Karin Wolters 1, Pei-Jing Pai 2 Yingwei Hu 2, Henry Lam 2, Rainer Bischoff 1

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1 9/23/ Chromosome 5 Peter Horvatovich 1, Karin Wolters 1, Pei-Jing Pai 2 Yingwei Hu 2, Henry Lam 2, Rainer Bischoff 1 1 University of Groningen, 2 Hong Kong University of Science and Technology Kyoto HUPO Initiative Assembly September 19, 2013

2 9/23/ Activities outline 1. Validation of eqtl studies 2. Glycopeptide identification workflow 3. Participation in QTAPAS experiment 4. C-HPP Wiki

3 1. Validation eqtl studies Transcription Factor RNA polymerase gene-coding region xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx DNA strand exon Promoter region xxxxxxxxxxxxxxxxxx Messenger RNA Coding SNP x Abnormal protein e.g. CFTR and α 1 -AT Deficiency Protein

4 1. Validation eqtl studies Transcription Factor RNA polymerase gene-coding region xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx exon DNA strand xxxxxxxxxxxxxxxxx Messenger RNA Promoter SNP Protein

5 1. Validation eqtl studies SNP X Cis-acting gene-coding region protein expression aa aa AA Trans-acting gene-coding region expression Cis- and Trans-acting SNPs aa aa AA

6 1. Validation eqtl studies Lung eqtls to Help Reveal the Molecular Underpinnings of Asthma, Hao et al., PLoS Genet Dec;8(12)

7 1. Validation eqtl studies Example of an eqtl: GSDMA (gasdermin)

8 1. Validation eqtl studies Example of an eqtl: GSDMA (gasdermin)

9 1. Validation eqtl studies Most asthma-associated SNPs control gene expression GSDM1 [17q21.1], GSDML [17q12] PNMT (phenylethanolamine N-methyltransferase) [17q12] CRKRS [17q12 ] PERLD1 (per1-like domain containing 1) [17q12] ORMDL3 [17q12] PPARBP (PPAR binding protein) [22q13.31] ERO1LB [1q42.3] CTNNA1 (cadherin-associated protein, alpha 1) [5q31.2] MYBPH [1q32.1] CHI3L1 [1q32.1] PCHD1 (Protocadherin-1) [5q31.3]

10 1. Validation eqtl studies Experimental Plan Develop SRM assays to quantify target proteins in tissue and lung-proximal body fluids. Develop targeted immunochemical assays (IHC, Western, ELISA). Perform functional studies in cell culture (cell lines & primary cells from patients). Make assays and reagents available to the research community

11 2. Glycosylation analysis by mass spectrometry Site-specific glycosylation profiling * Protease digestion * LC/MS Site 1 Site 2 * glycoprotein glycopeptide, peptide G 1 G 2 G 3 G 4 G 5 G 1 G 2 G 3 G 4 G 5 glycopeptide identification glycan profile on each glycosylation site

12 9/23/ Participation in QTAPAS experiment Collaboration with chromosome 16 (Juan Pablo Albar) QTAPAS: Quantitative Targeted Analysis in Proteomics. An Assessment Study Goal: Standardisation of protocols & sample distribution. Defining suitable standard samples to test, optimize and QC SRM/MRM analysis at EUPA laboratories contributing to HPP. Multicenter and multi-instrument assessment of SRM quantification in AQUA peptide spiked yeast sample: 6 Digested proteins, 25-fold concentration range in 3 concentration tiers, 2-3 labeled peptides/protein, L/H ratios

13 9/23/ Participation in QTAPAS experiment # Lab SRM MPM MS Instrument Flow rate LC Column Gradient time Scheduled 1 VHIO X 4000QTrap 300nL/min 75 µm x 15 cm 90 no 2 IACS X 4000QTrap 300 nl/min 75 µm x 15 cm 90 no 3 ABSciex X 5500QTrap 15 µl/min 500 µm x 10 cm 20 yes 4 INIBIC X 5500QTrap 300 nl/min 75 µm x 15 cm 100 no 5 UPF CRG X 5500QTrap 300 nl/min 75 µm x 12 cm 35 yes 6 CIMA X 5500QTrap 300 nl/min 75 µm x 15 cm 90 no 7 CNB X 5500Qtrap 300 nl/min 75 µm x 15 cm 90 no 8 UCM PCM X 5500QTrap 300 nl/min 75 µm x 15 cm 90 no 9 CSPI Lund X TSQ Vantage 300 nl/min 75 µm x 12 cm 60 yes 10 X TSQ Vantage 300 nl/min 75 µm x 15 cm 30 yes 11 Groningen X TSQ Vantage 300 nl/min 75 µm x 15 cm 60 yes 12 X TSQ Vantage 300 nl/min 75 µm x 15 cm 75 µm x yes 13 UV X 5600TripleTOF 300 nl/min cm 90 no 14 Navarra Bio. X 5500QTrap 300 nl/min 75 µm x 15 cm 90 no 15 Sigma X 5500QTrap 4 µl/min 500 µm x 15 cm 45 no 16 UA X Agilent nl/min 75 µm x 15 cm 90 yes 17 Turku X TSQVantage 300 nl/min 75 µm x 15 cm 90 yes 18 X 4000 QTrap 300 nl/min 75 µm x 15 cm 90 no UVIC 19 X Agilent nl/min 75 µm x 15 cm 90 no 20 UiB X 5500QTrap 250 µl/min 75 µm x 15 cm 90 no 21 EHU X Q Exactive 300 nl/min 50 µm x 15 cm IRBB X Synapt G1 250 nl/min 75 µm x 25 cm CIB X Orbitrap Velos 250 nl/min 75 µm x 15 cm CBMSO X Orbitrap Velos 300 nl/min 75 µm x 10 cm Salamanca X Orbitrap Velos 400 nl/min 75 µm x 10 cm LPCSIC UAB X Orbitrap Velos 0.8 µl/min 100 µm x 15 cm CCiT UB X Orbitrap Velos 400 nl/min 75 µm x 10 cm PCB X Orbitrap Velos 250 µl/min 75 µm x 25 cm CMU Geneva X Orbitrap Velos 250 µl/min 75 µm x 25 cm CICbioGUNE X Orbitrap XL ETD 300 nl/min 75 µm x 20 cm SRM data sets Mostly used MIAPE to submit results 10 MPM data sets

14 3. Participation in QTAPAS experiment Peak widths (example CAH1_GGPFSDSYR) 30-minute gradient 60-minute gradient 90-minute gradient Sample A

15 9/23/ C-HPP Wiki Peter Horvatovich (Chromosome 5), the Netherlands

16 9/23/ C-HPP Wiki Wiki workshop and activity at HUPO 2013 Upload or send me your posters and presenations that you made at HUPO related to C-HPP.

17 9/23/ Participants Peter Horvatovich Karin Wolters Rainer Bischoff Pei-Jing Pai, Yingwei Hu, Henry Lam,