Human plasma and peripheral blood mononuclear cell (PBMC) proteome databases for biomarker discovery studies in vivo [Elektronische Ressource] / Carolin Heim

Heim (J.L.)

Sujets

• Naturwissenschaften
• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 73
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Ernährungsphysiologie


Human plasma and peripheral blood mononuclear cell (PBMC) proteome databases for biomarker discovery
studies in vivo



Carolin Heim



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. D. Haller
Prüfer der Dissertation:
1. Univ.-Prof. Dr. H. Daniel
2. Univ.-Prof. Dr. J. J. Hauner

Die Dissertation wurde am 04.05.2010 bei der Technischen Universität München eingereicht und durch die
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 16.08.2010
angenommen.








































Table of Contents
Table of Contents

Zusammenfassung ................................................................................................................................... 1

Summary ................................................................................................................................................. 2

1. Introduction .................................................................................................................................... 3
1.1. Biomarker Discovery in Nutritional Research ....................................................................... 3
1.2. Impact of Dietary Protein Intake on Human Metabolism ...................................................... 4
1.3. Use of Proteomics Applications to Human Plasma and Blood Cells in Nutritional
Intervention Studies ............................................................................................................................ 7
1.4. The Plasma Proteome ............................................................................................................ 9
1.5. Proteome Analysis of Plasma and PBMC ............................................................................ 10
1.5.1. Sample Preparation ..................................................................................................... 11
1.5.2. Plasma Sample Processing .......................................................................................... 13
1.5.3. Protein Separation Techniques .................................................................................... 16
1.5.4. Protein Identification ................................................................................................... 19
1.5.5. Search Algorithms . 21
1.5.6. Plasma Protein Databases ............................................................................................ 21
1.5.7. The Sub-Proteomes of Human Plasma ........................................................................ 22
1.6. Identification of the Plasma Proteome ................................................................................. 23

2. Aim and Outline of the Thesis ...................................................................................................... 25

3. Materials and Methods ................................................................................................................. 26
3.1. Chemicals and Consumables ............................................................................................... 26
3.2. Instruments ........................................................................................................................... 26
3.3. The High-Protein Intervention Study ................................................................................... 26
3.3.1. Volunteer Recruiting ................................................................................................... 26
3.3.2. Study Design ............................................................................................................... 26
3.3.3. Sample Collection . 27
3.3.4. SDS Electrophoresis of the Protein Powder ................................................................ 27
3.4. Sample Preparation for Proteomics Analysis ....................................................................... 28
3.4.1. PBMC Preparation ...................................................................................................... 28
3.4.2. Plasma Pre-Fractionation ............................................................................................ 28
3.4.3. Protein Measurement ................................................................................................... 29
3.5. Electrophoretic Analysis of Plasma and PBMC .................................................................. 29
Table of Contents
3.5.1. Protein Separation Using 2D Gel Electrophoresis ...................................................... 29
st3.5.2. 1 Dimension ............................................................................................................... 30
nd3.5.3. 2 Dimension .............................................................................................................. 31
3.5.4. Staining of Gels ........................................................................................................... 31
3.5.5. Image Analysis ............................................................................................................ 32
3.6. Protein Identification ........................................................................................................... 32
3.6.1. Sample Preparation for MS Analysis .......................................................................... 32
3.6.2. MALDI Mass Spectrometry ........................................................................................ 33
3.6.3. Spectra Processing and Database Search .................................................................... 33
3.7. Analysis of Amino Acids and Clinical Parameters .............................................................. 33
3.7.1. Measurement of Amino Acids in Plasma and Urine ................................................... 33
3.7.2. Clinical Parameters in Plasma and Urine .................................................................... 33
3.8. Statistical Analysis ............................................................................................................... 34

4. Results and Discussion ............ 35
4.1. Method Development ........................................................................................................... 35
4.1.1. Sample Pre-Fractionation in Plasma ........................................................................... 35
4.1.2. Optimizing Electrophoresis for Plasma and PBMC .................................................... 36
4.2. 2DE Separation for Determining Within- and Between-Laboratories Variability .............. 40
4.3. In-House Proteome Databases for Human Plasma and PBMC Samples ............................. 41
4.3.1. The Plasma Database .................................................................................................. 41
4.3.2. The PBMC Database ................................................................................................... 45
4.4. A High-Protein Diet Human Intervention Study ................................................................. 49
4.4.1. Content of the Protein Powder .................................................................................... 49
4.4.2. Clinical Chemistry Parameters 51
4.4.3. Amino Acid Profiles during the Study Course ............................................................ 55
4.4.4. Analysis of the Plasma Proteome after Protein Supplementation ............................... 62
4.4.5. Analysis of the PBMC Proteome after ementation 69

5. General Remarks and Conclusions ............................................................................................... 74
5.1. Proteomics Applications in Human Intervention Studies .................................................... 74
5.2. The “Stability” of the Plasma and PBMC Proteomes .......................................................... 75
5.3. Application of 2D Maps of Plasma and PBMC in Biomarker Discovery ........................... 76
5.4. High-Protein Supplementation and Its Impact ..................................................................... 77
5.5. Future Perspectives .............................................................................................................. 78


Table of Contents
6. Appendix ...................................................................................................................................... 80
6.1. Abbreviations ....................................................................................................................... 80
6.2. Tables ................ 83

7. References ..............................................................................................