Global analysis of cellular protein dynamics by pulse-labeling and quanti tati ve mass spectrometry [Elektronische Ressource] / Björn Schwanhäußer. Gutachter: Thomas Sommer ; Matt hias Selbach ; Andreas Herrmann

Björn Schwanhäußer

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Global analysis of cellular protein dynamics by
pulse-labeling and quan ta ve mass spectrometry
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathema sch-Naturwissenscha lichen Fakultät I
der Humboldt-Universität zu Berlin
von
Dipl.-Biol. Björn Schwanhäußer
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies
Dekan der Mathema sch-Naturwissenscha lichen Fakultät I
Prof. Dr. Andreas Herrmann
Gutachter/innen: 1. Prof. Thomas Sommer
2. Prof. Ma hias Selbach
3. Prof. Andreas Herrmann
Tag der mündlichen Prüfung: 23.02.2010
tttitiftfttitifor LiamTable of contents
TABLE OF CONTENTS i
ABSTRACT v
ZUSAMMENFASSUNGi
ABBREVIATIONSii
I INTRODUCTION 12
I.1. Mass spectrometry in a nutshell 13
I.1.1. Instrumenta on and workfl ow of mass spectrometry 13
I.1.2. Quan ta ve approaches in mass spectrometry-based proteomics 17
I.1.2.1 Rela ve MS-based quan fi ca on with heavy stable-isotopes 17
I.1.2.2 Stable isotope labeling by amino acids in cell cultue (SILAC) 19
I.1.2.3 Absolute quan fi ca on of proteins 21
I.1.2.4 Label-free approaches 21
I.2. Biological implica ons of mass spectrometry-based quan ta ve proteomics 23
I.2.1. Transla onal regula on of gene expression 23
I.2.2. Regula on of gene expression exerted by microRNAs 23
I.2.3. The complex rela onship between mRNAs and proteins 25
I.3. Outline and objec ves of the thesis 29
II MATERIAL AND METHODS 30
II.1. General suppliers 30
II.2. General solu ons and bu ff ers 30
II.3. Solu ons and bu ff ers for LC-MS/MS sample prepara on and instrumenta on 31
II.4. Cell culture and prepara on of SILAC medium 31
II.4.1. e for pulsed SILAC (pSILAC) 31
II.4.2. Cell culture for determina on of mRNA and protein half-life 32
II.4.3. e for microRNA/LNA transfec on experiments 32
II.4.4. Cell culture for intensity based absolute quan fi ca on (iBAQ) 32
II.5. Prepara on of SILAC media 32
II.6. Quan fi ca on of luciferase expression by pulsed SILAC and luminescence 33
II.7. Treatment of HeLa cells with iron (FAC) or iron chelator (DFO) 34
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titititititititititititititititititititititititititititiII.8. microRNA or LNA transfec on and pulsed SILAC labeling 34
II.8.1. Synthe c miRNAs 35
II.8.2. Locked nucleic acids (LNAs) 35
II.9. Analysis of transfec on e ffi ciency 35
II.10. Mapping of protein and mRNA IDs and iden fi ca on of sequence mo fs
correlated with changes in protein produc on 36
II.10.1. Mapping 36
II.10.2. Iden fi ca on of correlated sequence mo fs 36
II.11. Genera on of luciferase reporter constructs 36
II.11.1. 3’ UTRs of the following genes were selected for cloning 37
II.11.2. Primers (5’-3’) 37
II.11.3. Polymerase chain reac on (PCR) 37
II.11.4. Agarose gel electrophoresis 37
II.11.5. Enzyma c DNA diges on 38
II.11.6. Liga on of DNA fragments 38
II.11.7. Prepara on of plasmid DNA 38
II.11.8. DNA Sequencing 38
II.12. Co-transfec on of synthe c miRNAs and 3’ UTR reporter constructs 38
II.13. Dual-Luciferase Assay 39
II.14. Immunoblo ng (Western Blo ng) 39
II.14.1. Valida on of puta ve miRNA targets 39
II.14.2. Valida on of protein half-lives 39
II.14.3. Detec on of proteins 40
II.15. Microarray data analysis 40
II.16. RNA isola on 41
II.17. Double pulse-labeling of NIH3T3 cells with heavy amino acids and 4-thiouridine 41
II.18. Prepara on of newly-synthesized and pre-exis ng RNA 41
II.18.1. Bio nyla on and purifi ca on of 4sU-labeled RNA 41
II.18.2. Separa on of bio nylated 4sU-RNA from unlabeled RNA 42
II.18.3. Determina on of separa on e ffi ciency of 4sU-labeled RNA 42
II.18.4. Es ma on of the doubling me of NIH3T3 cells 43
II.19. mRNA sample prepara on and sequencing 43
II.20. Calcula on of mRNA half-lives 43
II.21. Calcula on of absolute mRNA copy numbers 44
II.21.1. Correc on for di ff erent transcript mappabili es 45
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titititititititititititititititittitititititititititititititititititittitititititititiII.22. Quan ta ve real- me (qRT) PCR 45
II.23. Cycloheximide-chase analysis 47
II.24. Liquid-chromatography mass spectrometry (LC-MS) 47
II.24.1. In-gel diges on 47
II.24.2. In-solu on diges on 48
II.24.3. Prepara on of Stop and Go Extrac on Tips (StageTips) 48
II.24.4. HPLC and mass spectrometry 48
II.24.5. Processing of mass spectrometry data 50
II.25. Applica on of the Universal Proteomics Standard (UPS) 51
II.26. Calcula on of cellular protein copy numbers by intensity-based absolute
quan fi ca on (iBAQ) 51
II.27. Cluster analysis of gene ontology (GO) terms 51
II.28. Sta s cal analysis 52
II.29. Determina on of protein half-lives, response mes and mathema cal
modeling of gene expression 52
II.29.1. Calcula on of protein half-lives 52
II.29.2. Quan ta ve model of gene expression 53
II.29.3. Response mes 54
II.30. Energy calcula ons 55
II.31. Structural features a ff ec ng mRNA and protein stability 55
III RESULTS 56
III.1. Global analysis of cellular protein transla on by pulsed SILAC (pSILAC) 56
III.1.1. Establishment of a pulsed SILAC approach with two heavy-stable isotopes 56
III.1.2. pSILAC - as good as the tradi onals? 57
III.1.3. pSILAC accurately quan fi es over a high range of expression levels 59
III.1.4. Applying pSILAC to iron homoeastasis - a proof of principle 60
III.2. Widespread changes in protein synthesis induced by microRNAs 63
III.2.1. pSILAC quan fi es changes in protein produc on induced by miRNAs 63
III.2.2. Sequence characteris cs associated with reduced protein synthesis 65
III.2.3. miRNAs transla onally repress many direct target genes 67
III.2.4. An endogenous miRNA knock-down confi rms overexpression experiments 69
III.3. Genome-wide parallel quan fi ca on of mRNA and protein turnover and
levels in mammalian cells 71
III.3.1. Combined metabolic labeling of newly-synthesized RNA and proteins
enables de termina on of half-lives 71
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tititititititititititititititititititititititititititititititititiIII.3.1.1 Determina on of cellular protein half-lives 71
III.3.1.2 Determina on of cellular mRNA half-lives 75
III.3.2. Sequence characteris cs a ff ec ng mRNA and protein stability 76
III.3.3. Es ma ng cellular protein and mRNA levels 79
III.3.3.1 Absolute mRNA quan fi ca on 79
III.3.3.2 Absolute protein quan fi ca on by intensity-based absolute quan fi ca on
(iBAQ) 79
III.3.4. Correla on of protein and mRNA levels and half-lives 81
III.3.4.1 Half-lives substan ally determine gene expression kine cs 83
III.3.4.2 Half-lives op mize gene expression towards energy constraints 85
IV DISCUSSION 87
IV.1. Establishment of pulsed SILAC (pSILAC) 87
IV.2. The impact of microRNAs on the proteome 90
IV.3. Dynamic proper es of mammalian gene expression 94
V CONCLUSIONS AND OUTLOOK 101
VI REFERENCES 103
VII SUPPLEMENTARY INFORMATION 113
VII.1. Publica on list 113
VII.2. Awards 114
VII.3. Posters and talks 114
VII.4. Supplementary data 115
VII.5.1. Supplementary fi gures 115
VII.5.2. tary tables 119
VIII ACKNOWLEGDEMENTS 122
IX SELBSTÄNDIGKEITSERKLÄRUNG 123
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titititititititititititititititititiAbstract
Gene expression is a ghtly controlled process that is subject to transcrip onal and as post-
transcrip onal regula on. Current methods for system-wide gene expression analysis detect changes in
mRNA abundance but neglect regula on at the level of transla on. The fi rst part of the thesis therefore
describes the establishment of modifi ed version of the classic SILAC (stable isotope labeling by amino
acids in cell culture) approach, that is rou nely used in quan ta ve mass spectrometry to assay
rela ve changes in protein levels. However, since steady-state protein levels refl ect the net outcome
of antagonizing protein synthesis and protein degrada on processes, the SILAC method cannot be
harnessed to explicitly measure di ff erences in protein transla on. In contrast, in the newly-devised
approach termed pulsed SILAC (pSILAC) di ff eren ally treated cell popula ons are simultaneously
transferred to culture medium supplemented with di ff erent versions of stable-isotope labeled heavy
amino acids. This is advantageous over the classic SILAC strategy as mass spectrometry-based rela ve
quan fi ca on is exclusively based on the newly-synthesized heavy protein amounts. This enables the
specifi c detec on of di ff erences in protein transla on resul ng from the di ff eren al treatment. The
second part of the thesis presents the applica on of the pSILAC approach to globally quan fy the
impact of small, non-coding RNAs (microRNAs) as major players in post-transcrip onal regula on onto
the proteome. Intriguingly, ectopic over-expression or knock-down of a single microRNA both a ff ected
protein produc on of hundreds of proteins. Notably, pSILAC iden fi ed several target genes as exclusively
transla onally regulated since changes in corresponding transcript levels measured in parallel by
conven onal DNA microarrays were virtually absent. Recording newly-synthesized protein amounts
with heavy amino acids in a pulsed-labeling approach has also been used to determi