Structural characterisation of protein derived N-glycans by mass spectrometry: application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2001
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Münster, Univ., Diss., 2001 |
| Beschreibung: | VII, 157 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Structural characterisation of protein derived N-glycans by mass spectrometry |b application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation |c vorgelegt von Dijana Šagi |
| 246 | 1 | |a Structural characterisation of protein-derived N-glycans by mass spectrometry | |
| 264 | 1 | |c 2001 | |
| 300 | |a VII, 157 S. |b Ill., graph. Darst. | ||
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| 500 | |a Münster, Univ., Diss., 2001 | ||
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Datensatz im Suchindex
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CONTENTS
_I
CONTENTS
ABBREVIATIONS.
V
1 INTRODUCTION. 1
1.1 MASS SPECTROMETRY IN ANALYTICAL BIOCHEMISTRY. 1
1.1.1 IMPORTANT TERMS IN MASS SPECTROMETRY. 1
1.1.2 INSTRUMENTATION. 2
1.1.2.1 ION SOURCE. 3
1.1.2.1.1 MATRIX-ASSISTED LASER DESORPTION/IONISATION (MALDI). 3
1.1.2.1.2 ELECTROSPRAY IONISATION (ESI). 4
1.1.2.2 MASS ANALYSERS.-. 7
1.1.2.2.1 TIME-OF-FLIGHT (TOF). 7
1.1.2.2.2 QUADRUPOLE MASS ANALYSER. 10
1.1.2.3 TANDEM MASS SPECTROMETRY AND CID. 13
1.2 N-GLYCOSYLATIONS OF PROTEINS.:. 14
1.2.1 BIOSYNTHESES OF N-LINKED OLIGOSACCHARIDES. 15
1.2.2 FUNCTION OF N-LINKED OLIGOSACCHARIDES. 19
1.2.3 E-SELECTIN AND E-SELECTIN LIGAND-1. 20
1.2.4 CONGENITAL DISORDERS OF GLYCOSYLATION (CDG). 24
1.2.4.1 DIAGNOSIS OF CDG-TYPES. 25
1.2.5 STRUCTURAL ANALYSIS QF N-GLYCANS. 27
1.2.5.1 DETECTION OF N-GLYCOSYLATION. 27
1.2.5.2 RELEASE OF N-GLYCANS. 27
1.2.5.3 CHROMATOGRAPHIC PROFILING OF RELEASED N-GLYCANS. 28
1.2.5.4 MONOSACCHARIDE COMPOSITION ANALYSIS.
/.
. 30
1.2.5.5 METHYLATION ANALYSIS. 30
1.2.5.6 NUCLEAR MAGNETIC RESONANCE (NMR). 31
1.2.5.7 SEQUENTIAL EXOGLYCOSIDASE DIGESTION. 31
1.2.5.8 MASS SPECTROMETRY. 31
2 OBJECTIVES OF THE STUDY.
35
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/964761831
CONTENTS
_N
3 EXPERIMENTAL PART. 37
3.1 IN-SOLUTION DEGLYCOSYLATION. 37
3.2 IN-GEL DEGLYCOSYLATION. 38
3.2.1 SODIUMDODECYLSULPHATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE). 38
3.2.2 PROTEIN REDUCTION AND ALKYLATION. 39
3.2.3 ENZYMATIC RELEASE OF N-GLYCANS IN-GEL. 39
3.2.4 EXTRACTION OF N-GLYCANS AFTER IN-GEL DEGLYCOSYLATION. 39
3.3 DESALTING OF NATIVE N-GLYCANS. 40
3.4 METHYLATION OF SIALIC ACIDS. 40
3.5 DESALTING OF METHYLATED N-GLYCANS. 40
3.6 ENZYMATIC REMOVAL OF SIALIC ACIDS. 41
3.7 IN-GEL TRYPTIC DIGESTION. 41
3.7.1 PEPTIDE EXTRACTION AND PURIFICATION. 41
3.8 MASS SPECTROMETRY. 42
3.8.1 MALDITOF MS. 42
3.8.2 ESI Q-TOF MS. 45
3.9 HPAEC/PAD. 47
3.10 STANDARD N-GLYCANS. 49
4 RESULTS AND DISCUSSION. 51
4.1 METHODS FOR STRUCTURAL ANALYSIS AND SEQUENCING OF N-GLYCANS. 51
4.1.1 N-DEGLYCOSYLATION OF PROTEINS. 51
4.1.1.1 IN-SOLUTION DEGLYCOSYLATION OF PROTEIN MIXTURE. 52
4.1.1.2 IN-GEL DEGLYCOSYLATION OF PROTEIN COMPONENTS FROM THE
MIXTURES. 52
4.1.2 PURIFICATION OF THE RELEASED N-GLYCANS. 53
4.1.3 METHYLATION OF SIALIC ACIDS. 55
4.1.4 MS ANALYSIS. 58
4.1.4.1 N-GLYCANS PROFILING BY MALDI MS. 58
4.1.4.1.1 NEUTRAL N-GLYCANS. 58
4.1.4.1.2 SIALYLATED N-GLYCANS AND MIXTURES OF SIALYLATED AND
NEUTRAL N-GLYCANS. 59
4.1.4.1.3 METHYLATED SIALYLATED N-GLYCANS. 63
CONTENTS
4.1.4.1.4 QUANTITATIVE ASPECTS OF MALDI MS OF N-GLYCANS. 63
4.1.4.2 N-GLYCAN ANALYSIS BY ESI Q-TOF MS. 59
4.1.4.2.1 SEQUENCING OF NEUTRAL N-GLYCANS BY (+)ESI Q-TOF
MS. 65
4.1.4.2.2 SEQUENCING OF SIALYLATED N-GLYCANS (-)ESI Q-TOF
MS.*. 72
4.2 STRUCTURAL DETERMINATION OF N-GLYCANS OF E-SELECTIN LIGAND-1
GLYCOPROTEIN. 82
4.2.1 ANALYSIS OF ESL-1 MYC PROTEIN EXPRESSED IN CHO-PRO'5 CELLS. 82
4.2.1.1 IDENTIFICATION OF THE PROTEIN SPECIES IN THE ADDITIONAL MAJOR
BAND IN SDS-PAGE. 84
4.2.1.2 ANALYSIS OF N-GLYCANS AFTER IN-SOLUTION DEGLYCOSYLATION. 84
4.2.1.2.1 NATIVE N-GLYCAN FIXTURE.,T. 85
4.2.1.2.2 NEUTRAL AND METHYLATED SIALYLATED N-GLYCAN MIXTURE. 90
4.2.1.3 ANALYSIS OF N-GLYCANS AFTER IN-GEL DEGLYCOSYLATION. 92
4.2.2 ANALYSIS OF ESL-1 MYC PROTEIN EXPRESSED IN CHO-DHFR' CELLS. 94
4.2.2.1 ANALYSIS OF N-GLYCANS AFTER IN-SOLUTION DEGLYCOSYLATION. 94
4.2.2.1.1 NATIVE N-GLYCAN MIXTURE. 94
4.2.2.1.2 NEUTRAL AND METHYLATED SIALYLATED N-GLYCAN MIXTURE. 98
4.2.2.2 ANALYSIS OF N-GLYCANS AFTER IN-GEL DEGLYCOSYLATION.100
4.2.2.3 ESI MS/MS ANALYSIS.102
4.3 ABERRANT N-GLYCOSYLATION IN CONGENITAL DISORDERS OF
GLYCOSYLATION.106
4.3.1 CLINICAL PHENOTYPE OF THE CDG PATIENT.106
4.3.2 ISOELECTRIC FOCUSING OF SERUM TRANSFERRIN.107
4.3.3 N-GLYCOSYLATION OF SERUM TRANSFERRIN.107
4.3.4 N-GLYCOSYLATION OF SERUM AL-ANTITRYPSIN.114
4.3.5 N-GLYCOSYLATION OF PLASMA PROTEINS.
T.116
4.3.5.1 ANALYSIS OF MIXTURE OF NEUTRAL AND SIALYLATED N-GLYCANS.116
4.3.5.1.1 MALDI MS.117
4.3.5.1.2 HPAEC/PAD.118
4.3.5.2 ANALYSIS OF DESIALYLATED N-GLYCANS.123
4.3.5.2.1 MALDI MS.123
4.3.5.2.2 HPAEC/PAD. 125
133
5 SUMMARY.
CONTENTS
IV
6 REFERENCES. 139
7 ACKNOWLEDGEMENTS. 153
8 PUBLICATIONS AND PRESENTATIONS. 155
9 LEBENSLAUF. 157 |
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| illustrated | Illustrated |
| indexdate | 2024-10-09T18:04:50Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-009820392 |
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| owner_facet | DE-91 DE-BY-TUM DE-29T DE-355 DE-BY-UBR |
| physical | VII, 157 S. Ill., graph. Darst. |
| publishDate | 2001 |
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| spelling | Šagi, Dijana Verfasser aut Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation vorgelegt von Dijana Šagi Structural characterisation of protein-derived N-glycans by mass spectrometry 2001 VII, 157 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Münster, Univ., Diss., 2001 Massenspektrometrie (DE-588)4037882-2 gnd rswk-swf Glucane (DE-588)4157662-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Glucane (DE-588)4157662-7 s Massenspektrometrie (DE-588)4037882-2 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009820392&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Šagi, Dijana Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation Massenspektrometrie (DE-588)4037882-2 gnd Glucane (DE-588)4157662-7 gnd |
| subject_GND | (DE-588)4037882-2 (DE-588)4157662-7 (DE-588)4113937-9 |
| title | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation |
| title_alt | Structural characterisation of protein-derived N-glycans by mass spectrometry |
| title_auth | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation |
| title_exact_search | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation |
| title_full | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation vorgelegt von Dijana Šagi |
| title_fullStr | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation vorgelegt von Dijana Šagi |
| title_full_unstemmed | Structural characterisation of protein derived N-glycans by mass spectrometry application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation vorgelegt von Dijana Šagi |
| title_short | Structural characterisation of protein derived N-glycans by mass spectrometry |
| title_sort | structural characterisation of protein derived n glycans by mass spectrometry application to glycosylation analysis of e selectin ligand 1 and potentials in diagnosis of congenital disorders of glycosylation |
| title_sub | application to glycosylation analysis of E-selectin ligand-1 and potentials in diagnosis of congenital disorders of glycosylation |
| topic | Massenspektrometrie (DE-588)4037882-2 gnd Glucane (DE-588)4157662-7 gnd |
| topic_facet | Massenspektrometrie Glucane Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009820392&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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