Enrichment and Identification of Methylation at the Proteome Level Alexandra Star A thesis ...

228 Pages·2015·5.27 MB·English

Enrichment and Identification of Methylation at the Proteome Level

Alexandra Star

A thesis submitted to the
Faculty of Graduate and Postdoctoral Studies
in partial fulfillment of the requirements for the
Degree of Master of Science in Biochemistry

Department of Biochemistry, Microbiology, and Immunology
Faculty of Medicine
University of Ottawa

©Alexandra Star, Ottawa, Canada, 2016
ABSTRACT
Methylation is a post-translational modification which occurs on lysine and arginine
residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our
laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and
arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled
with mass spectrometry.
Simplifying a lysate with subcellular fractionation prior to enrichment increased the
identification of methylation sites by 39.5% while using multiple proteases for digestion
increased identification by 27%. Combining these methods yielded a 47.2% increase.
Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169
sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database.
This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and
can be a key tool in the field of methylation allowing for the enrichment and identification of
methylated proteins.

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ACKNOWLEDGEMENTS
I would like to thank Dr. Daniel Figeys for giving me the opportunity to study under
his guidance and the guidance of Dr. Jean-François Couture. Over the past two years I have
learned about proteomics, mass spectrometry, and the difficulties in studying post-
translational modifications like methylation. Dr. Figeys gave me the opportunity to be
independent, and for that I am grateful.
I would also like to acknowledge my thesis advisory committee members Dr.
Alexandre Blais and Dr. Jocelyn Côté for their guidance and re-direction in my project.
I would also like to thank Deeptee Seebun for training me in the lab. I would like to
acknowledge Dr. Janice Mayne for motivating and supporting me throughout my masters
and for meeting with me weekly to discuss experiment ideas and working tirelessly to edit all
of my TAC reports, presentations and my thesis itself. I am grateful to Dr. Zhibin Ning
whose method has made my project possible and for all of his discussion and scientific
advice. Although we work differently, he has helped me change the way I approach
problems and has challenged me throughout my learning process. Lastly I would like to
thank Dr. Sylvain Lanouette for the many discussions in regards to methylation and SMYD2,
and for the guidance in writing my thesis
I would also like to thank my lab mates Deeptee Seebun, Shelley Deeke, Dr.
Amanda Starr, Myriam Cramet, Kiara Chu, Dr. Bo Xu, Dr. Zhibin Ning, Dr. Janice Mayne,
Dr. Kerwin Chiang, Dr. Rui Chen, Dr. Xu Zhang, and Jasmine Moore for all the motivation,
scientific discussion, and friendship over the course of my two years in the lab.
I would also like to thank Ngoc Vuong for his friendship and support during my
undergraduate thesis at Health Canada, and the mentorship of Dr. Renaud Vincent which has
inspired me to pursue the path of scientific research.
Finally, I would like to thank my friends, family and Matt. Their support and
encouragement helped me get through the joys and the hardships both in life and during the
course of my studies.

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STATEMENT OF CONTRIBUTION
All experiments were conducted by Alexandra Star (AS). Experimental design was
conducted by Daniel Figeys (DF), Janice Mayne (JM) and Zhibin Ning (ZN) and AS. Data
analysis was conducted by AS with the advice and mentoring of ZN.

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TABLE OF CONTENTS
ABSTRACT .......................................................................................................................................... ii
ACKNOWLEDGEMENTS .................................................................................................................. iii
STATEMENT OF CONTRIBUTION ................................................................................................. iv
ABBREVIATIONS ............................................................................................................................ viii
LIST OF FIGURES .............................................................................................................................. xi
LIST OF TABLES ............................................................................................................................... xii
LIST OF APPENDICES ..................................................................................................................... xii
1. INTRODUCTION ............................................................................................................................. 1
I. Proteomics and Post-Translational Modifications.......................................................................... 1
II. Protein Methylation ...................................................................................................................... 2
i. Arginine Methylation ................................................................................................................. 3
ii. Lysine Methylation .................................................................................................................... 4
III. Biological Roles of Protein Methylation ..................................................................................... 7
i. Protein Methylation in Transcriptional Regulation .................................................................... 7
ii. Methylation in mRNA splicing ................................................................................................. 9
iii. Effect of Methylation on Protein Stability ............................................................................. 10
IV. Protein Methylation in Disease ................................................................................................. 11
i. Cancer ....................................................................................................................................... 11
ii. Embryo Development .............................................................................................................. 12
iii. Methylation in Vaccination .................................................................................................... 13
iv. Cardiovascular Disease ........................................................................................................... 14
v. Spinal Muscular Atrophy ......................................................................................................... 14
V. Current Approaches to Studying Methylation ............................................................................ 15
i. Low-Throughput Targeted Approaches ................................................................................... 15
ii. High-Throughput Enrichment-Based Screening ..................................................................... 15
iii. Methyl-Lysine Binding Domains to Enrich for Methylated Proteins .................................... 16
iv. Pan-Methyl Antibodies to Enrich for Methylated Peptides .................................................... 16
v. Derivatization Coupled with Affinity Enrichment .................................................................. 17
vi. Click Chemistry to Determine Methyltransferase Targets ..................................................... 17
vii. Predictive Modeling of Methylation Sites ............................................................................. 18
VI. Novel Approach to Enrich for Methylated Peptides ................................................................. 18
VII. Rationale .................................................................................................................................. 20
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VIII. Statement of Hypothesis and Objectives ................................................................................ 21
i. Hypothesis ................................................................................................................................ 21
ii. Objectives ................................................................................................................................ 21
2. MATERIALS AND METHODS .................................................................................................... 26
I. Cell Culture .................................................................................................................................. 26
II. Heavy-Methionine Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) ............ 26
III. Cell Lysis ................................................................................................................................... 27
IV. Subcellular Fractionation........................................................................................................... 27
V. Immunoblotting .......................................................................................................................... 28
VI. Proteomics Sample Preparation ................................................................................................. 29
i. Protein Precipitation ................................................................................................................. 29
ii. In-solution Protein Digestion .................................................................................................. 29
iii. Peptide Desalting .................................................................................................................... 30
iv. Synthesis of 1,1,3,3-tetraisopropoxypropane (TiPP) .............................................................. 31
v. Preparation of Phthaldialdehyde (OPA) Reagent .................................................................... 31
vi. ProMENADe Enrichment Reaction ....................................................................................... 31
VII. LC-MS/MS Measurement ........................................................................................................ 32
VIII. Mascot Database Search ......................................................................................................... 33
IX. Data Analysis ............................................................................................................................ 34
i. Proteomics Tool Suite .............................................................................................................. 34
ii. Statistical Analysis .................................................................................................................. 34
iii. Gene Ontology Analysis ........................................................................................................ 35
iv. SILAC Incorporation Calculation........................................................................................... 35
v. Protein Abundance Across Organisms Database (PaxDb) Analysis ....................................... 35
3. RESULTS ........................................................................................................................................ 37
I. Optimization of the ProMENADe Protocol in Biological Samples ............................................. 37
i. Determination of Optimal Amount of Protein Starting Material .............................................. 37
ii. Discussion of Optimal Amount of Protein Starting Material .................................................. 38
iii. ProMENADe Reaction Identifies More Confident Methylation Sites ................................... 41
iv. Discussion for ProMENADe Reaction Identifies More Confident Methylation Sites ........... 45
II. Simplification of the Protein Mixture to Increase MS Identification.......................................... 52
i. Subcellular Fractionation to Increase Methylation Site Identification by MS/MS ................... 58
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ii. Discussion for Subcellular Fractionation to Increase Methylation Site Identification by
MS/MS ......................................................................................................................................... 62
iii. Digestion with Multiple Proteases to Increase Methylation Site Identification by MS/MS .. 65
iv. Discussion for Digestion with Multiple Proteases to Increase Methylation Site Identification
by MS/MS .................................................................................................................................... 72
v. Comparison of Tryptically-Digested Whole Cell Lysate to Nuclear and Cytosolic Fractions
Digested by Trypsin, Chymotrypsin and Glu-C .......................................................................... 73
vi. Discussion for the Comparison of Tryptically-Digested Whole Cell Lysate to Nuclear and
Cytosolic Fractions Digested by Trypsin, Chymotrypsin and Glu-C .......................................... 82
III. Heavy Methionine Labeling in Cell Culture to Reliably Identify Methylated Peptides ............ 83
i. Discussion ................................................................................................................................. 84
4. CONCLUSIONS AND FUTURE DIRECTIONS .......................................................................... 87
REFERENCES .................................................................................................................................... 92
APPENDICES ................................................................................................................................... 104
Appendix 1: Supplementary Results ............................................................................................. 104
Appendix 2: List of methylation sites identified by MS/MS in ranging amounts of starting material
from 0.2 mg to 10 mg. ................................................................................................................... 106
Appendix 3: List of methylation sites identified by MS/MS in a ProMENADe-enriched and non-
enriched samples. ........................................................................................................................... 130
Appendix 4: List of methylation sites identified by MS/MS in starting material, cytosolic and
nuclear fractions. ........................................................................................................................... 172
Appendix 5: List of methylation sites identified by MS/MS in tryptic, chymotryptic and Glu-C
digested samples. ........................................................................................................................... 187
Appendix 6: List of methylation sites identified by MS/MS in a tryptically digested whole cell
lysate compared to cytosolic and nuclear fractions digested with trypsin, chymotrypsin, and Glu-C.
....................................................................................................................................................... 199
Appendix 7: Curriculum vitae for Alexandra Star. ........................................................................ 212

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ABBREVIATIONS
ACN: acetonitrile
ADAM10: a disintegrin and metalloproteinase domain-containing protein 10
ADMA: asymmetric di-methylation of arginine
ADP: adenosine diphosphate
ANOVA : analysis of variance
Blimp1: PR domain zinc-finger protein
BRG1: Brahma-related gene1, also known as transcription activator BRG1 or ATP-
dependent helicase SMARCA4
CA150: coactivator 150 kDa
CARM1: coactivator-associated arginine methyltransferase
CBP: CREB-binding protein
CDK: cyclin-dependent kinase
Clr4: Histone lysine-N-methyltransferase, histone H3 lysine-9 specific
COMPASS: Complex of Proteins Associated with Set1
CREB: cAMP response element-binding protein
DAVID: The Database for Annotation, Visualization and Integrated Discovery
DDAH: dimethylarginine dimethylaminohydralase
DMEM: Dulbecco’s Modified Eagle’s Medium
DNA: deoxyribonucleic acid
DNMT: DNA methyltransferase
DOT1L: DOT1-like histone H3K79 methyltransferase
DTT: dithiothreitol
E2F: transcription factor E2F
E2F1: transcription factor E2F1
EEF1A1: elongation factor 1 A 1
EHMT1: histone-lysine-N-methyltransferase EHMT1, also known as GLP1
EHMT2: histone-lysine-N-methyltransferase EHMT2, also known as G9a
ERα: estrogen receptor alpha
ESCC: esophageal squamous cell carcinoma
EZ/E(z): enhancer of zeste
FA: formic acid
FBS: fetal bovine serum
FDR: false discovery rate
G9a: see EHMT2
GLP1: see EHMT1
GO: Gene Ontology
GRAVY: grand average of hydropathy
H3K4: histone H3 at lysine 4
H3K9: histone H3 at lysine 9
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H3K36: histone H3 lysine 36
H3K79: histone H3 lysine 79
H3R8: histone H3 at arginine 8
H4R3: histone H4 at arginine 3
HBHA: heparin-binding hemagglutinin
hBRM: human Brahma protein
HCD: high energy C-trap dissociation
HCT116: human colon colorectal cell line
HCl: hydrochloric acid
HEK: human embryonic kidney
HeLaS3: a human cervix adenocarcinoma cell line
HILIC: hydrophilic interaction liquid chromatography
HP1: heterochromatin protein 1
HP1β: heterochromatin protein 1 β
HPLC: high performance liquid chromatography
HRP: horseradish peroxidase
IAA: iodoacetamide
KMTs: lysine methyltransferases
L3MBTL1: lethal (3) malignant brain tumour like protein 1
LBP: lipopolysaccharide binding protein
LC: liquid chromatography
3XMBT: 3 malignant brain tumour repeats
MDA: malondialdehyde
MEFs: mouse embryonic fibroblasts
MMA: mono-methylation on arginine
mmu: milli mass unit
mRNA: messenger RNA
MS: mass spectrometry
MYND: Myeloid, Nervy, and DEAF-1
MYPT1: myosin phosphatase target subunit 1
m/z: mass to charge ratio
NaOH: sodium hydroxide
NFĸB: nuclear factor kappa B
NO: nitric oxide
NOS: nitric oxide synthase
OmpB: outer membrane protein B
OPA: ortho-phthaldialdehyde
p21 : cyclin-dependent kinase inhibitor
p53: tumour protein p53
p300: histone acetyltransferase p300
PaxDb: Protein Abundance Across Organisms Database
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PBS: phosphate buffered saline
pI: isoelectric points
ppm: parts per million
pRb: tumour suppressor retinoblastoma protein
PRMTs: protein arginine methyltransferases
ProMENADe: Protein Methylation Enrichment by Neutralizing Amine through
Derivatization
PTM: post translational modification
REAP: Rapid Efficient and Practical
RelA: REL-associated protein, nuclear factor kappa B p65 subunit
RIZ: retinoblastoma-interacting zinc-finger
RNA: ribonucleic acid
SAH: S-adenosyl-L-homocysteine
SAM: S-adenosyl-L-methionine
SAP49: RNA binding protein SAP49
SCX: strong cation exchange
SDMA: symmetric di-methylation of arginine
SDS: sodium dodecyl sulphate
SET: Suppressor of variegation 3-9, Enhancer of zeste, and Trithorax
SET1A/B: SET-domain protein 1 A/B
SET7/9: SET-domain protein 7/9
SILAC: stable isotope labeling by amino acids in cell culture
siRNA: small interfering RNA
SMA: spinal muscular atrophy
SMN: survival of motor neuron
SMYD: SET and MYND domain-containing protein
Snail: zinc-finger protein SNAI1
snRNA: small nuclear ribonucleic acid
snRNP: small nuclear ribonucleic protein
SRC3: steroid-receptor coactivator-3
STAT3: signal transducer and activator of transcription 3
SUV39: suppressor of variegation 3-9
Suv39h1: human homolog of SUV39
SUV4-20: suppressor of variegation 4-20
Swi6: chromatin-associated protein Swi6
TAF10: TBP-associated factor TAF10
TBP: TATA-box binding protein
TBST: tris-buffered saline and tween
THW loop: threonine-histidine/tryptophan loop
TiPP: 1,1,3,3-tetraisopropoxypropane
Trx: trithorax
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