1 / 18

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

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

magar
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related