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glycosylation analysis of gel separated proteins proteomics 2001 1 350 361 n.
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Glycosylation analysis of gel-separated proteins (Proteomics 2001, 1, 350-361)

Glycosylation analysis of gel-separated proteins (Proteomics 2001, 1, 350-361)

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Glycosylation analysis of gel-separated proteins (Proteomics 2001, 1, 350-361)

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  1. Glycosylation analysis of gel-separated proteins(Proteomics 2001, 1, 350-361) 생명과학부 박사 2학기 2001-31228 하 병 집

  2. Contents 1. Introduction 2. Why gel electrophoresis? 3. A short history of methods for the preparation and detection of glycopeptides and glycans from gel-separated glycoproteins 4. Global glycosylation profiling 5. Site-specific glycosylation profiling 6. Glycan sequencing 6.1 Exoglycosidase sequencing 6.2 MS/MS 7. Further perspectives 8. Concluding remarks

  3. Complexity of glycan structure ; information-rich molecule that guide many biological processes ? 1)different monosaccharide can join to each other through any of several hydroxyl groups and different linkage 2) C-1 linkage can have either αor βconfiguration 3) Extensive branching is possible

  4. 2. Why gel electrophoresis? • Gel-separated protein analysis의 장점: • 따로 purification이 필요 없다. (small quantity protein, large membrane protein의 경우 중요) • Reducing 1-D SDS-PAGE는 protein subunit들을 분리시켜줌 • Loss 위험 없이 여러 가지 in-gel manipulations 실험 가능 • Glycoprotein analysis 에서 2-DE의 한계 • 1-DE is generally favored over 2-DE for glycoprotein separation • Very poorly resolved trains of spots or even smears on 2-DE gels • Many glycoproteins are large and membrane bound;2-DE 어려움

  5. 3. Preparation and detection methods on gel-separated glycoproteins • A short history of methods • 1990, Monosaccharide composition analysis: PVDF, acid hydrolysis • 1992, Hydrazinolysis release of glycans after gel electrophoresis (Radiolabeling, fluorescent HPLC detection) • 1993, Sequential endoglycosidases release of N-linked glycans from PVDF membrane (HPAEC/PAD detection) • 1996, Oligosaccharide compositional analysis from 2-DE separation • 1997, MALDI-MS and fluorescent HPLC to profile and sequence glycans (50-100 pmol of protein applied to the gel) • 1998, ESI-MS profiles of in-gel released glycans

  6. A good variety of methods are available • 1-DE / 2-DE • Direct gel format / blots on membrane • PNGase F / Hydrazinolysis release of N-glycans (PNGase A in plant and insect glycoproteins) • O-glycosidase / reductive β-elimination release of O-glycans • HPAEC-PAD / HPLC with fluorescent labeling / MS detection • Complementary use of MS and HPLC • MS : prior derivatization 불필요 Standard molecule 없이 분자량만으로 isobaric monosaccharide composition 추정 가능(Rapid screening of the glycans present) Isobaric species 구별 못함 • HPLC : easy monosaccharide composition analysis

  7. 4. Global glycosylation profiling ; Analysis of all released N-glycans of a gel-separated glycoprotein using MALDI-MS • sample의 overall complexity 평가 가능 • next detailed analysis의 방향 제시 • MALDI profiles say relative quantities of oligosaccharides in a mixture (relative ionization efficiencies 매우 유사) • Can be performed rapidly on many samples in parallel • Glycosylation profile의 변화 관찰 시 유용 (fig.1)

  8. 5. Site-specific glycosylation profiling ; more complete information on glycoprotein heterogeneity • Fig2A와 fig2D의 비교 M/z 3400-5000 peaks 없어지고, 여러 개의 새로운 peak 나타남 MS/MS sequencing of the peptide(m/z 1288.8): glycosylation site CVYN(QK)S Mass differences of glycosylated/non- species: biantennary complex type with NeuAc/Gc (partial exoglycosidase digests in B and C) • Peptide mass mapping with and without prior enzymatic deglycosylation :potential for high throughput/high sensitivity screening for the N-glycosylation • Glycopeptides generally produce weaker signal : peak를 인지 못하기 쉽다. • Exact site can be determined by performing the deglycosylation in 50% 18O- labeled water(2 Da spacing, fig 3)

  9. 6. Glycan sequencing Full characterization includes monosaccharide composition, sequence, branching, linkage and anomericity analysis 6.1 Exoglycosidase sequencing • Detection of exoglycosidase reaction products by MS or HPLC • Commonly used glycosidases for sequencing (Table 3) • Sequential treatment with single enzymes or glycan pool divided into several portions and treated with arrays of enzymes in parallel(in 1 day) • Quick and convenient, but depends on the enzyme specificity

  10. 6.2 MS/MS • MS/MS review of glycan structure determination : Proteomics 2000, 1, 311-328 • MALDI-PSD(post-source decay) analysis(fig 4) B- and Y-type ions : sequence and branching information Internal fragment ions : respective monosaccharide composition information

  11. 7. Further perspectives • PTM analysis의 sensitivity and specificity 높이려는 연구 필요 ESI-Quadrupole TOF, MALDI quadrupole TOF MALDI PSD 보다 sensitivity, resolution 매우 우수 • Sample preparation 방법에서의 개선점 Glycans sample must be cleaned extensively prior to MS analysis Graphitized carbon resin binds oligosaccharide but not salts and other contaminants • Nonglycosylated peptides often greatly hampers the detection and analysis of the glycopeptide in MALDI or ESI MS ‘fish out’ glycopeptides with ConA lectin-coupled resin

  12. 8. Concluding remarks • Protein identification (minimal sequence information이면 충분)과 PTM analysis(full sequence coverage 필요)의 근본적 차이 • Glycoform heterogeneity의 특성상, 더 높은 specificity와 sensitivity가진 분석법 필요함( carbohydrates의 primary structure는 peptide에 비해 수십, 수백 배 복잡) • Carbohydrate analysis는 이제 막 긴 여정을 시작했어 대, 아직 proteomics의 주류에 끼지 못함