1 SUMMARY ...................................................... 1
1 ZUSAMMENFASSUNG .............................................. 3
2 INTRODUCTION ................................................. 5
2.1 The spliceosome ......................................... 6
2.1.1 U snRNP particles are the submits of the
spliceosome ...................................... 8
2.1.2 Assembly of snRNPs .............................. 11
2.1.3 Mechanism of splicing and assembly of the
spliceosome ..................................... 13
2.2 Mass spectrometry ...................................... 16
2.2.1 Matrix assisted laser desorption ionization
(MALD1) mass spectrometry ....................... 17
2.2.2 Electro spray ionization (ESI) mass
spectrometry .................................... 22
2.2.3 Sequencing of peptides and nucleic acids ........ 27
2.3 Chromatography in protein research ..................... 32
2.4 Introduction 3D structure analysts of macromolecular
complexes using EM ..................................... 38
2.5 Analysis of protein-protein and protein-RNA
interactions based on MS ............................... 41
2.5.1 Analysis of protein-protein interactions by
crosslinking and mass spectrometry .............. 42
2.5.2 Analysis of protein-RNA interactions by
crosslinking and mass spectrometry .............. 43
2.6 Aim of this work ....................................... 46
3 MATERIALS AND METHODS ....................................... 49
3.1 Materials .............................................. 49
3.1.1 Chemicals, fine chemicals, general materials .... 49
3.1.2 Enzymes and Enzyme inhibitors ................... 50
3.1.3 Chromatography and mass spectrometry
equipment ....................................... 50
3.1.4 General Laboratory materials .................... 51
3.1.5 Instruments ..................................... 51
3.2 Methods ................................................ 52
3.2.1 Purification of spliceosomal complexes .......... 52
3.2.1.1 Cultivation of HeLa cells and
purification of small nuclear
ribonucleoprotein complexes (snRN Ps)
from nuclear extracts .................. 52
3 2 1.2 Purification of 12S Ul snRNPs and 12S
U2 snRNPs for UV-crosslinking
studies ................................ 53
3.2.1.3 Preparation of the spliceosomal A-,
B- and C-complexes ..................... 54
3.2.2 UV crosslinking experiments ..................... 55
3.2.2.1 UV-irradiation and subsequent sample
processing for semi-preparative scale
purification of peptide- RNA
heteroconjugates ....................... 55
3.2.2 2 Semi-preparative purification of
UV-induced peptide-RNA
heteroconjugates ....................... 56
3.2.2.3 Small analytical scale generation of
UV-induced peptide-RNA
heteroconjugates ....................... 57
3.2 2.4 UV-crosslinking and subsequent sample
processing of spliceosomal complexes ... 58
3.2.2.5 Phosphatase treatment removes
phosphopeptides prior to the ТiO2
enrichment ............................. 58
3 2.2.6 Microscale offline ТiO2 enrichment for
crosslinks and phosphopeptides ......... 58
3.2.2.7 Offline Immobilized Metal Ion
Affinity Chromatography (IMAC)
enrichment ............................. 59
3.2.2.8 Offline ТiO2 enrichment for
crosslinks and phophopeptides to test
suitable conditions for the online
2D-enrichment setup .................... 59
3.2.2.9 2D-online enrichment for crosslinks
and phosphopeptides - setup and
workflow of the 2D-chromatography
system ................................. 60
3.2.2.10 MALDI analysis of 1D- or 2D nanoLC
purified peptide-RNA
heteroconjugates ....................... 62
3.2.2.11 LC-online ESI-MSMS analysis of ТiO2-
purified peptide-RNA
heteroconjugates ....................... 62
3.2.2.12 Data analysis strategy for large
crosslink data sets generated from
ESI-MSMS ............................... 62
3.2.2.13 UV-Crosslinking of RNA oligomers and
peptides ............................... 63
3.2.3 Chemical modifications probing the solvent
accessibility in native particles ............... 64
3.2.3.1 Alkylation of cysteines using
iodacetamide ........................... 64
3.2.3.2 Modification of lysines using di-m
ethy 1 -am mo-bor ane complexes ........ 65
3.2.4 Electron Carbon Film Assisted Digest (ECAD)
directly correlates EM and MS ................... 66
3.2.4.1 General remark on the preparation of
glutaraldehyde fixed protein
complexes .............................. 66
3.2 4.2 Reconstitution of GroEL(14)GroES(7)-
2ATP complexes for ECAD experiments .... 66
3.2.4.3 Preparation of U1 snRNPs for
adsorption onto carbon films ........... 67
3 2 4.4 Preparation of U1 snRNPs in complex
with snurportin nuclear import
factor ................................. 67
3.2.4.5 Preparation of 25S [U4/U6.U5] tri-sn
RNPs for adsorption onto carbon
films .................................. 67
3 2 4.6 Preparation of high salt U5 snRNPs
for adsorption onto carbon films ....... 68
3.2.4.7 Preparation of spliceosomal
B-complexes for adsorption onto
carbon films ........................... 68
3.2.4.8 Preparation of electron microscopy
carbon films and sample preparation
for EM-image recording ................. 68
3.2.4.9 Electron microscopy .................... 69
3.2.4.10 Sample preparation of the Electron
Carbon Film Assisted Digest (ECAD)
and the in-parallel in-solution
digestion .............................. 70
3.2.4.11 Offline LC-MALDI-TOF/TOF and online-
ESI-MSMS analysis for ECAD ............. 71
3.2.4.12 Data analysis strategy for peptide
detection and evaluation of data ....... 71
3.2.4.13 Relative quantification of 18O/16O
labeled peptides ....................... 72
3.2.4.14 Generation of color-coded 3D models .... 72
3.2.5 General methods for molecular biology and
biochemistry .................................... 73
3.2.5.1 Phenol-chlorophorm-isoamyl extraction
for separation of RNA and protein and
subsequent ethanol or acetone
precipitations ......................... 73
3.2.5.2 Denaturing poly aery lamide gel
electrophoresis for separation of
RNA .................................... 73
3.2.5.3 Determination of protein
concentration in aqueous solution
(Bradford-Assay) ....................... 74
3.2.5.4 Denaturing SDS-polyacrylamide gel
electrophoresis for separation of
proteins ............................... 74
3.2.6 General chromatography methods ....................... 75
3.2.6.1 Size exclusion chromatography (SEC) .... 75
3.2.6.2 Semi-preparative reversed phase
liquid chromatography (RP-LC) .......... 76
3.2.6.3 Nano-scale reversed phase liquid
chromatography - setup and operation ... 76
3.2.6.4 Offline desalting using centrifuge
microspin columns ...................... 78
3.2.7 Mass Spectrometry methods ....................... 79
3.2.7.1 In-gel hydrolysis of proteins .......... 79
3.2.7.2 In-solution hydrolysis of proteins ..... 80
3.2.7.3 In-solution hydrolysis of RNA .......... 81
3.2.7.4 Thin-layer preparation with α-cyano-
4-cinnamic acid ........................ 81
3.2.7.5 Dried droplet preparation with
diverse matrices ....................... 81
3.2.7.6 Instrumental parameters for the MALDI
measurements ........................... 82
3.2.7.7 Instrumental parameters for the ESI
measurements ........................... 83
4 RESULTS ..................................................... 84
4.1 Analysis of protein-RNA interactions in spliceosomal
complexes .............................................. 84
4.1.1 Miniaturization of the chromatographic
separation using 1D nano-LC ..................... 86
4.1.2 Optimizing enzymatic digests of the snRNPs and
evaluation by 1D nano-LC ........................ 87
4.1.3 Evaluation of crosslink enrichment, MS based
data generation and automated data analysis
strategies ...................................... 90
4.1.3.1 Enrichment of crosslinks using
titanium dioxide nano-columns
incorporated to a online-2D nano
liquid chromatography interfaced with
MALDI-TOF/TOF .......................... 90
4.1.3.2 Offline enrichment of crosslinks from
crude mixtures using titanium dioxide
micro columns interfaced with
ESI-MSMS used for high-throughput
analysis ............................... 99
4.1.3.3 Evaluation of a peptide-RNA crosslink
prediction workflow by a restricted
database search using highest mass
accuracy and RNA marker ions alone .... 104
4.1.4 Crosslinking of native U1 snRNPs, offline
TiO2 enrichment and ESI-MSMS .................... 106
4.1.5 Crosslinking of 12S U2 snRNPs. offline TiO2-
enrichment and ESI-MSMS ........................ 114
4.1.6 Detection of direct interactions of amino
acids and oligonucleotides by probing the
chemical reactivity ............................ 117
4.1.6.1 Protection of cysteine residues in
native U1 and U2 snRNPs from
alkylation with iodacetamide .......... 119
4.1.6.2 Protection of lysine residues
in native U1 and U2 snRNPs from
di-methylation with a di-methyl-
amino-borane complex .................. 121
4.2 Analysis of the protein-protein network in
spliceosomal complexes ................................ 124
4.2.1 Improved digest quality of an integrative
sample preparation workflow for MS using
glutaraldehyde - Electron Carbon Film
Assisted Digest (ECAD) ......................... 124
4.2.2 Peptides are generated with higher
reproducibility from ECAD as compared to
in-solution digest - example of a multi-copy
protein complex GroEL14-GroES7 ................. 134
4.2.3 Probing solvent accessibility of lysine
residues in Ul snRNPs by applying
glutaraldehyde fixation and electron carbon
film assisted digest (ECAD) .................... 136
4.2.4 Comparison of the lysine reactivity towards
glutaraldehyde (ECAD) and the trimethyl-
amino-borane complex in Ul snRNPs .............. 140
4.2.5 Probing solvent accessibility of lysine
residues in [U4/U6.U5] tri-snRNP applying
glutaraldehyde jixation and electron carbon
film assisted digest (ECAD) .................... 143
4.2.6 ECAD enables the detection
ofsubstochiometrically bound factors to
[U4/U6.U5] tri-snRNP due to detection of
interacting peptide regions .................... 148
4.2.7 ECAD enables the detection
of substochiometrically bound factors to U1
snRNP due to detection of interacting
peptide regions ................................ 151
4.2.8 Correlation between structure and
composition using quantitative ECAD is
suitable for defining a stable core
particle ....................................... 153
5 DISCUSSION ................................................. 158
5.1 Protein-RNA interactions in spliceosomal
complexes ............................................. 158
5.1.1 Phosphate specific enrichment of peptide-
RNA heteroconjugates from crude mixtures
by ТiO2 enables higher sensitivity by
reduction of purification steps ................ 160
5.1.2 Impact of the sample purification on the
data analysis workflow ......................... 165
5.1.3 De-novo identified crosslinks in native U1
snRNPs correlate with other protein
interaction studies ............................ 167
5.1.4 De-novo identified crosslinks in 12S U2
snRNPs propose a crucial role of cysteines
in Sm Core assembly and depict requirement
for more RNA sequence .......................... 171
5.1.5 Unusual cro.sslinking chemistry observed
for cysteines can be explained by oxidation
of nucleobases during UV-irradiation ........... 172
5.1.6 Improvements for future UV crosslinking
studies ........................................ 174
5.2 Protein-protein interactions in spliceosomal
complexes ............................................. 175
5.2.1 Protection of e-amines of lysines can be
analyzed by ECAD or di-methyl-amino-borane
modification - validation of the method ........ 176
5.2.2 Implications of ECAD far the interactions
within the [U4/U6.U5] tri-snRNP ................ 179
5.2.3 Defining stably bound and loosely
associated protein constituents of the
protein complexes improved upon ECAD sample
processing ..................................... 181
5.2.4 General implications of ECAD on the
analysis of macromolecular assemblies, and
outlook for further applications ............... 185
6 REFERENCES .................................................. 188
7 APPENDIX .................................................... 207
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