Research Collection Doctoral Thesis Analysis of the beta-cell N-glyco surface proteome using label- free quantitative mass spectrometry, and targeted candidate biomarker validation in mouse models of type 2 diabetes Author(s): Danzer, Carsten Friedrich Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-005940078 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH N° 18564 Analysis of the beta-cell N-glyco surface proteome using label-free quantitative mass spectrometry, and targeted candidate biomarker validation in mouse models of type 2 diabetes. Submitted to ETH ZURICH for the degree of DOCTOR OF SCIENCES by CARSTEN FRIEDRICH DANZER Diploma-Biologist, University of Konstanz Date of birth 21. September 1977 Citizen of Germany accepted on the recommendation of Professor Dr. Wilhelm Krek Professor Dr. Claes Wollheim Professor Dr. Ruedi Aebersold 2009 Table of contents Table of contents 1. Summary ............................................................................................................................... 4 2. Zusammenfassung: ............................................................................................................... 6 3. Abbreviations: ....................................................................................................................... 8 4. Introduction ........................................................................................................................ 11 4.1. The pancreatic beta-cell ................................................................................................. 11 4.1.1. The beta-cell in the islet of Langerhans ................................................................... 11 4.1.2. Environmental influences on insulin secretion and beta-cell metabolism ............... 13 4.2. Diabetes mellitus ............................................................................................................ 15 4.2.1. Classifications of diabetes mellitus .......................................................................... 15 4.2.2. Genetic susceptibility ............................................................................................... 17 4.3. Animal models of disease ............................................................................................... 18 4.3.1. Current rodent models in the study of beta-cell function ........................................ 18 4.3.2. C57BL/6J High fat diet mouse model ..................................................................... 18 4.3.3. Leptin deficient ob/ob mouse model (Lep ob/ob).................................................... 19 4.3.4. Leptin receptor deficient db/db mouse model (db/db mice) .................................... 20 4.3.5. ZDF rat model (fa/fa rats) ........................................................................................ 20 4.4. Proteomics ...................................................................................................................... 21 4.4.1. Proteome-wide expression analysis ......................................................................... 21 4.4.2. Membrane proteomics ............................................................................................. 22 4.4.3. Quantitative proteome analysis ................................................................................ 23 4.4.4. Labeling techniques for quantification .................................................................... 24 4.4.5. Label-free quantification .......................................................................................... 25 4.4.6. Absolute quantification ............................................................................................ 26 4.4.7. Proteomics studies with pancreatic islets and beta-cells .......................................... 26 5. Results .................................................................................................................................. 28 5.1. MIN6 Cell Surface Capturing catalogue ....................................................................... 28 5.2. MIN6 Whole Cell Capturing catalogue ......................................................................... 30 5.3. Human islets Whole Cell Capturing catalogue .............................................................. 31 1 Table of contents 5.4. Islet/beta-cell N-glyco protein catalogue ....................................................................... 32 5.5. Technical and biological variation ................................................................................ 34 5.6. Quantitative CSC of MIN6 cells stimulated with 25 mM D-glucose ............................. 34 5.7. Quantitative CSC of MIN6 cells stimulated with 25 mM D-glucose plus 20 nM GLP-1 ............................................................................................................................................... 38 5.8. Beta-cell biomarker studies ............................................................................................ 43 5.9. Diet induced obesity (DIO) mouse model blood analysis .............................................. 44 5.10. ob/ob mouse model blood analysis .............................................................................. 48 5.11. Results from shotgun approach with DIO plasma samples ......................................... 52 5.12. Results from MRM approach in DIO and ob/ob mouse models .................................. 53 6. Discussion ............................................................................................................................ 62 6.1. The N-glyco proteomes of beta-cells and islets .............................................................. 62 6.2. Surface protein dynamics under glucose treatment ....................................................... 63 6.3. Surface protein dynamics under glucose in combination with GLP-1 treatment .......... 70 6.4. Candidate Biomarker evaluation in mouse models of T2D disease .............................. 81 7. Material and Methods ........................................................................................................ 88 7.1. Animal models ................................................................................................................ 88 7.2. Mouse blood plasma sampling ....................................................................................... 88 7.3. Mouse blood plasma analysis ........................................................................................ 88 7.4. Intraperitoneal Glucose Tolerance Testing (IPGTT) .................................................... 88 7.5. Mass Spectrometry ......................................................................................................... 88 7.6. LC-MS/MS-Analysis ....................................................................................................... 89 7.7. Database search ............................................................................................................. 90 7.8. Cell Surface Capturing (CSC) ....................................................................................... 91 7.9. Whole Cell Capturing (WCC) ........................................................................................ 92 7.10. Directed LC-MS/MS ..................................................................................................... 92 7.11. Label-free Quantification ............................................................................................. 93 2 Table of contents 7.12. N-glycosite blood sample processing ........................................................................... 93 7.13. Reference Peptides ....................................................................................................... 94 7.14. Peptide Separation ....................................................................................................... 94 7.15. Off-gel electrophoresis ................................................................................................. 94 7.16. Chemicals ..................................................................................................................... 94 7.17. Cell lines and culture conditions .................................................................................. 95 7.18. Human Pancreatic Islets .............................................................................................. 95 7.19. Insulin secretion assay and insulin content ................................................................. 95 7.20. Q-PCR .......................................................................................................................... 95 7.21. Bioinformatics analysis ................................................................................................ 96 7.22. Primer pairs ................................................................................................................. 96 7.23. Statistics ....................................................................................................................... 97 8. Acknowledgement ............................................................................................................... 98 9. References: .......................................................................................................................