1 Introduction ................................................. 1
1.1 Aim ..................................................... 2
1.2 Structure of thesis ..................................... 2
2 Proteomics background ........................................ 4
2.1 Quantitative proteomics within current biology .......... 4
2.2 Quantitative techniques orthogonal to mass
spectrometry ............................................ 6
2.2.1 Gel electrophoresis .............................. 6
2.2.2 Western blots .................................... 7
2.3 Sample preparation prior to mass spectrometry ........... 7
2.3.1 Protein separation using sodium dodecyl sulfate
Polyacrylamide gel electrophoresis (SDS-PAGE) .... 7
2.3.2 Peptide separation using reversed phase liquid
chromatography ................................... 8
2.4 Mass spectrometry of peptides ........................... 9
2.4.1 Orbitrap mass spectrometer ....................... 9
2.4.2 Data-dependent acquisition ...................... 10
2.4.3 Fragmentation of peptides ....................... 11
2.4.4 Peptide identification from fragment spectra .... 12
2.5 Quantitative mass spectrometry ......................... 12
2.5.1 Quantification based on stable isotopic
labelled peptides ............................... 13
2.5.1.1 Heavy oxygen labelling of peptides
upon enzymatic cleavage ................ 13
2.5.1.2 Chemical labelling using ICAT .......... 14
2.5.1.3 Chemical labelling using iTRAQ ......... 14
2.5.1.4 Stable isotopic labelling by amino
acids in cell culture (SILAC) .......... 15
2.5.1.5 Absolute quantification of proteins
(AQUA) ................................. 16
2.5.1.6 Absolute quantification of proteins
using QCAT ............................. 16
2.5.2 Label-free quantification ........................ 17
2.5.2.1 Definition of a quantitative
"feature" ............................... 17
2.5.2.2 General label-free quantification work
flow .................................... 19
2.5.2.3 SuperHirn ............................... 20
2.5.2.4 Remaining challenges .................... 21
3 The label-free pipeline ..................................... 23
3.1 Overview of processing steps ........................... 23
3.2 Algorithmic improvements ............................... 24
3.2.1 Loading of pep.xml files into SuperHirn ......... 24
3.2.2 Improving SuperHirn's feature alignment ......... 25
3.2.3 Annotation of quantitative features with
peptide identifications ......................... 28
3.2.4 Improving SuperHirn memory management ........... 28
3.2.5 Improving SuperHirn usability ................... 29
3.3 Statistical procedures ................................. 30
3.3.1 False discovery rate (FDR) of peptide
identification .................................. 30
3.3.2 Data normalization .............................. 31
3.3.2.1 Normalization to standard peptides ..... 31
3.3.2.2 Log transformation ..................... 32
3.3.2.3 Central tendency normalization ......... 33
3.3.3 Evaluation of protein-level quantitative
changes ......................................... 33
4 Assessing label-free quantification performance ............. 36
4.1 Model mixtures of standard proteins .................... 36
4.2 Assessing the platform capabilities .................... 37
4.2.1 Linear dynamic range ............................ 37
4.2.2 Duplicate reproducibility ....................... 37
4.3 From quantifying peptides to quantifying proteins ...... 39
4.4 Testing the effects of varying LC gradient lengths ..... 40
4.5 Performance comparison of label-free software
programs ............................................... 42
4.5.1 Software usability .............................. 42
4.5.2 Result quality .................................. 43
5 Biological applications ..................................... 46
5.1 Adenomatous polyposis coli (APC) gene knockout in Mus
musculus liver ......................................... 46
5.1.1 Introduction .................................... 46
5.1.2 Proteomic results overview ...................... 47
5.1.3 Biological validation of label-free
quantification pipeline ......................... 47
5.1.4 Comparing label-free quantification with 2D
gel electrophoresis ............................. 48
5.1.5 Perturbation of energy metabolism in APC-KO
mice ............................................ 49
5.2 Dehydration survival of dauer state Caenorhabditis
elegans ................................................ 51
5.2.1 Introduction .................................... 51
5.2.2 Desiccation of worms for proteomic analysis ..... 52
5.2.3 Proteomic results ............................... 53
5.3 Somitogenesis in Danio Rerio ........................... 56
5.3.1 Introduction .................................... 56
5.3.2 Preparation of zebrafish embryos for mass
spectrometry .................................... 56
5.3.3 Proteomic results ............................... 57
6 Perspectives for quantitative inference ..................... 65
6.1 Theoretical design ..................................... 65
6.2 Inference of zebrafish somitogenesis proteins .......... 67
7 Concluding remarks .......................................... 68
7.1 Conclusions ............................................ 68
7.2 Future directions ...................................... 69
8 Materials and methods ....................................... 70
8.1 Reagents ............................................... 70
8.2 Peptide and protein standards .......................... 70
8.3 Preparation of standard protein model protein
mixtures ............................................... 71
8.4 Preparation of zebrafish samples ....................... 71
8.5 Preparation of Mouse brain samples ..................... 71
8.6 Preparation of APC knockout mice ....................... 71
8.7 Extraction and gel separation of APC-KO mouse
proteins ............................................... 72
8.8 Preparation of C. elegans samples ...................... 72
8.9 LC-MS/MS analyses ...................................... 72
8.10 Data processing ........................................ 73
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