Proteomics and post-translational modification studies in patients with multiple sclerosis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 115 S. Ill., graph. Darst. |
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| adam_text | PROTEOMICS AND POST-TRANSLATIONAL
MODIFICATION STUDIES IN PATIENTS WITH MULTIPLE
SCLEROSIS
DISSERTATION
DER MATHEMATISCH-NATURWISSENSCHAFTLICHEN FAKULTAT
DER EBERTIARD KARLS UNIVERSITAT TUBINGEN
ZUR ERLANGUNG DES GRADES EINES
DOKTORS DER NATURWISSENSCHAFTEN
(DR. RER. NAT.)
VORGELEGT VON
BUSHRA AMIN
AUS KARACHI, PAKISTAN
TUBINGEN
2014
HTTP://D-NB.INFO/106490081X
TABLE OF CONTENTS
1. ZUSAMMENFASSUNG 1
2. SUMMARY 3
ACKNOWLEDGEMENTS 9
ABBREVIATIONS 11
LIST OF FIGURES 15
LIST OF TABLES 17
3. INTRODUCTION 19
3.1. MULTIPLE SCLEROSIS 19
3.1.1.
CLINICAL FEATURES 19
3.1.2.
PATHOPHYSIOLOGY
21
3.1.3. ETIOLOGY 22
3.1.4.
CURRENT DIAGNOSIS 22
3.2. BIOMARKERS IN MULTIPLE SCLEROSIS 23
3.3. PROTEOMICS STUDIES IN MULTIPLE SCLEROSIS 24
3.4. POST-TRANSLATIONAL MODIFICATION STUDIES IN MSD 25
3.4.1.
CITRULLINATION OR DEIMINATION 26
3.4.2.
PEPTIDYL ARGININE DEIMINASE: CITRULLINATION CATALYZING ENZYME 27
3.4.2.1.
ISOZYMES OF PAD 27
3.4.3. PATHOPHYSIOLOGICAL
ASPECTS OF PAD ENZYME AND
CITRULLINATION 29
3.4.3.1. CITRULLINATION IN HUMAN PHYSIOLOGY 30
3.4.3.2. CITRULLINATION AND PAD
IN HUMAN PATHOLOGY 31
3.5. AIM OF THIS WORK 33
4. MATERIALS AND METHODS 35
4.1. SAMPLE PREPARATION 35
4.1.1.
SAMPLING AND
HANDLING CRITERIA 35
4.1.2.
DEPLETION OF ABUNDANT SEROLOGICAL PROTEINS 35
4.1.2.1. ALBUMIN DEPLETION 35
4.1.2.2. IMMUNOGLOBULIN DEPLETION 36
4.1.2.3. DEPLETION OF 14 MOST-ABUNDANT PROTEINS 36
4.1.3.
PROTEIN QUANTIFICATION 36
4.2. PROTEIN SEPARATION 36
4.2.1. SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 36
4.2.1.1. ONE-DIMENSIONAL POLYACRYLAMIDE
GEL ELECTROPHORESIS (ID-PAGE) 36
4.2.1.2. TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS (2D-PAGE) 36
4.2.1.3. TWO-DIMENSIONAL
DIFFERENTIAL GEL
ELECTROPHORESIS (2D-DIGE) 37
4.2.1.3.1. FLUORESCENT DYE-LABELING 37
4.2.1.3.2. FOCUSING AND SECOND
DIMENSION 37
4.2.1.3.3. DIFFERENTIAL IMAGE ANALYSIS (DIA) 37
4.3. VISUALIZATION OF PROTEINS 38
4.3.1. COOMASSIE STAINING 38
4.3.2.
SILVER STAINING 38
4.3.3. REVERSIBLE PONCEAU S STAINING 38
4.4. EXTRACTION, ISOLATION AND PURIFICATION
OF PROTEINS 38
4.4.1.
EXTRACTION OF KERATIN FROM CORNIFIED SKIN TISSUES 38
4.4.2.
ISOLATION OF PAD FROM RABBIT SKELETAL MUSCLES 39
4.4.2.1. TISSUE
HOMOGENIZATION 39
4.4.2.2. PRE
FRACTIONATION 39
4.4.2.3. ION-EXCHANGE
CHROMATOGRAPHY 39
PAGE | 5
4.4.2.4. AFFINITY PURIFICATION OF PAD 39
4.4.3.
ISOLATION OF MYELIN BASIC PROTEIN (MBP) 39
4.4.3.1. ACID EXTRACTION METHOD 39
4.4.3.2. CHLOROFORM EXTRACTION METHOD 40
4.4.3.3. SEC-BUTANOL
EXTRACTION METHOD 40
4.4.4.
ISOLATION OF MYELINOLIGODENDROCYTE GLYCOPROTEIN (MOG) 40
4.4.4.1. EXTRACTION
OF MYELIN FROM SUBCELLULAR FRACTIONS 40
4.4.4.2. SEPERATION OF BASIC PROTEIN 40
4.5. IMMUNOASSAYS 40
4.5.1. WESTERN
BLOTS 40
4.5.2. ENZYME-LINKED
IMMUNOSORBENT ASSAY (ELISA) 42
4.6. ANALYTICAL CHEMISTRY 43
4.6.1.
REVERSE PHASE HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC) 43
4.6.2.
MALDI-TOF-MS 43
4.6.2.1. SAMPLE DESALTING 43
4.7. GENERATION OF ANTIBODIES 43
4.7.1. ANTI-MODIFIED CITRULLINE ANTIBODIES 43
4.7.2.
ANTI-CITRULLINE ANTIBODIES 44
4.8. IN VITRO CITRULLINATION OF PROTEINS 45
4.9. IN VITRO CARBAMYLATION OF PROTEINS 45
4.10.LMMOBILIZATION OF PROTEINS 45
4.11.MODIFICATION OF PROTEIN/PEPTIDE-BOUND CITRULLINE 45
4.11.1.2,3-BUTANEDIONE (BMO) AND ANTIPYRINE (AP) MODIFICATION 45
4.11.2.HYDROXYPHENYLGLYOXAL MODIFICATION 46
4.12.PAD ACTIVITY
ASSAY 46
4.12.1.COLORIMETRIC ASSAY 46
4.12.2.
MASS SPECTROMETRY-BASED ASSAY 46
4.13.PROTEOMIC
S 46
4.13.1.LN-GEL DIGESTION AND PEPTIDE MASS FINGERPRINTING 46
4.13.2.0ENATURATION OF SERUM PROTEINS AND IN-SOLUTION DIGESTION 47
4.13.3.
NANOLC-MS/MS 47
4.14.SYNTHESIS OF CITRULLINE-REACTIVE BEADS (CRBS) 48
4.14.1.FUNCTIONALIZATION OF RESIN WITH ETHYLENEDIAMINE 48
4.14.2.COUPLING OF 4-HYDROXYMETHYLBENZOIC ACID (HMB) 49
4.14.3.LNTRODUCTION OF BASE-LABILE ESTER BOND 49
4.14.4.BEADS FUNCTIONALIZATION WITH HYDROXYPHENYLGLYOXAL 49
4.15.SPECIFIC ENRICHMENT OF CITRULLINATED PEPTIDES 49
4.15.1.
ENRICHMENT WITH CITRULLINE-REACTIVE BEADS (CRBS) 49
4.15.2.
IMMUNOPRECIPITATION (IP) OF CITRULLINATED PEPTIDES 50
4.16.AUTOANTIBODIES DETECTION 50
5. RESULTS AND
DISCUSSION 51
5.1. IDENTIFICATION OF MSCL-RELATED PROTEINS 51
5.1.1.
SERUM PROTEOME ANALYSIS 51
5.1.1.1. DEPLETION OF ABUNDANT PROTEINS 52
5.1.2. DIFFERENTIAL PROTEOME EXPRESSION PROFILING 53
5.1.3.
IDENTIFICATION OF PROTEINS BY PEPTIDE MASS FINGERPRINTING (PMF) 56
5.1.3.1. NANOLC-MS/MS
ANALYSIS 59
5.2. VALIDATION OF CANDIDATE BIOMARKERS 59
5.2.1.
SEMI-QUANTITATIVE VALIDATION OF IDENTIFIED BIOMARKERS 59
5.2.2. QUANTITATIVE VALIDATION OF PROPOSED BIOMARKERS 61
5.3. EXTRACTION AND PURIFICATION OF PEPTIDYLARGININE DEIMINASE 64
PAGE | 6
5.3.1.
PREFRACTIONATION OF PAD 65
5.3.2. WEAK
ANION-EXCHANGE CHROMATOGRAPHY 65
5.3.3. AFFINITY PURIFICATION OF PAD 65
5.3.4.
PAD ACTIVITY
DETECTION ASSAY 67
5.3.4.1. COLOUR DEVELOPMENT REAGENT (COLDER) ASSAY 67
5.3.4.2. MASS
SPECTROMETRY-BASED ACTIVITY ASSAY 68
5.3.5.
SEQUENCE ANALYSIS OF PAD BY PEPTIDE
MASS FINGERPRINTING 68
5.4. IDENTIFICATION OF MSCL-RELATED CITRULLINATED PROTEINS 70
5.4.1.
ANTI-MODIFIED CITRULLINE ANTIBODIES (AMC) 70
5.4.1.1. GEL-BASED DETECTION OF MSCL-RELATED CITRULLINATED PROTEINS 71
5.4.1.2. IN VITRO HOMO-CITRULLINATION: AN INTRUSION ARTIFACT 74
5.4.2.
MONOCLONAL ANTI-CITRULLINE ANTIBODIES 75
5.4.3.
POLYCLONAL ANTI-CITRULLINE ANTIBODIES (PAB) 76
5.5. GEL-FREE DETECTION OF CITRULLINATED PROTEINS/PEPTIDES 77
5.5.1.
ENRICHMENT OF MODIFIED CITRULLINATED PEPTIDES BY IMMUNOPRECIPITATION
(IP) WITH
ANTI-MODIFIED CITRULLINE ANTIBODIES 78
5.5.2.
MASS SPECTROMETRY-BASED IDENTIFICATION OF CITRULLINATED
PROTEINS/PEPTIDES 79
5.5.2.1. MODIFICATION OF UREIDO GROUP WITH BUTANEDIONE AND ANTIPYRINE 80
5.5.2.2.
GLYOXAL-BASED MODIFICATION OF CITRULLINE 81
5.5.3.
SPECIFIC ENRICHMENT OF CITRULLINATED PEPTIDES WITH CITRULLINE-REACTIVE
BEADS (CRBS). 84
5.5.3.1. ENRICHMENT OF CITRULLINATED PEPTIDES FROM IN WTRO-CITRULLINATED
BSA 85
5.5.3.2. ENRICHMENT OF CITRULLINATED PEPTIDES FROM MYELIN BASIC PROTEIN
(MBP) 88
5.5.3.3. ENRICHMENT OF CITRULLINATED PEPTIDES FROM SERA OF MSD PATIENTS
90
5.6. EXTRACTION OF MYELIN BASIC PROTEIN (MBP) 94
5.7. EXTRACTION OF MYELIN OLIGODENDROCYTE GLYCOPROTEIN (MOG) 95
5.8. EXTRACTION OF KERATIN FROM CORNIFIED LAYER OF HUMAN EPIDERMIS 96
5.9. PROPOSAL FOR A CLINICAL ASSAY FOR
MSD: AUTO ANTIBODIES-BASED DETECTION 97
6. BIBLIOGRAPHY 99
7. CONTRIBUTIONS 112
8. AUTHOR S SCIENTIFIC/GROUP CONTRIBUTIONS 113
8.1. OPTIMIZATION OF IN-GEL DIGESTION PROTOCOL 113
8.2. LARGE-FORMAT 2DE SET-UP
AND PROTEOMICS SUMMER SCHOOL
AND HAND-ON WORKSHOP
ATTENDANCE AND 113
8.3. PROTEOMICS SERVICES 113
8.4. TEACHING ASSISTANCE AND MENTORING 113
8.5. CONFERENCE/MEETINGS CONTRIBUTIONS 114
8.6. PUBLICATIONS BY THE AUTHOR 114
CURRICULUM VITAE
PAGE | 7
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| spelling | Amin, Bushra 1982- Verfasser (DE-588)1063985994 aut Proteomics and post-translational modification studies in patients with multiple sclerosis vorgelegt von Bushra Amin 2014 115 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Tübingen, Univ., Diss., 2014 Proteomanalyse (DE-588)4596545-6 gnd rswk-swf Multiple Sklerose (DE-588)4040693-3 gnd rswk-swf Posttranslationale Änderung (DE-588)4204069-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Multiple Sklerose (DE-588)4040693-3 s Proteomanalyse (DE-588)4596545-6 s Posttranslationale Änderung (DE-588)4204069-3 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027815980&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Amin, Bushra 1982- Proteomics and post-translational modification studies in patients with multiple sclerosis Proteomanalyse (DE-588)4596545-6 gnd Multiple Sklerose (DE-588)4040693-3 gnd Posttranslationale Änderung (DE-588)4204069-3 gnd |
| subject_GND | (DE-588)4596545-6 (DE-588)4040693-3 (DE-588)4204069-3 (DE-588)4113937-9 |
| title | Proteomics and post-translational modification studies in patients with multiple sclerosis |
| title_auth | Proteomics and post-translational modification studies in patients with multiple sclerosis |
| title_exact_search | Proteomics and post-translational modification studies in patients with multiple sclerosis |
| title_full | Proteomics and post-translational modification studies in patients with multiple sclerosis vorgelegt von Bushra Amin |
| title_fullStr | Proteomics and post-translational modification studies in patients with multiple sclerosis vorgelegt von Bushra Amin |
| title_full_unstemmed | Proteomics and post-translational modification studies in patients with multiple sclerosis vorgelegt von Bushra Amin |
| title_short | Proteomics and post-translational modification studies in patients with multiple sclerosis |
| title_sort | proteomics and post translational modification studies in patients with multiple sclerosis |
| topic | Proteomanalyse (DE-588)4596545-6 gnd Multiple Sklerose (DE-588)4040693-3 gnd Posttranslationale Änderung (DE-588)4204069-3 gnd |
| topic_facet | Proteomanalyse Multiple Sklerose Posttranslationale Änderung Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027815980&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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