1 / 20

Ara

Glycosyl Composition Analysis. Gal. Inositol. Ara. Peak Area. Gal. Ara. Gal. Gal. Glc. Man. Xyl. Glc. 10. 15. 20. 25. 30. Time (minutes). Glycosyl Linkage Analysis. 3,6-Gal. t-Ara(f). 3-Gal. Intensity. 6-Gal. 4-Gal. 4-Glc. t-Gal. Inositol. t-Man. 5-Ara(f). t-Glc.

adamsf
Télécharger la présentation

Ara

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. Glycosyl Composition Analysis Gal Inositol Ara Peak Area Gal Ara Gal Gal Glc Man Xyl Glc 10 15 20 25 30 Time (minutes)

  2. Glycosyl Linkage Analysis 3,6-Gal t-Ara(f) 3-Gal Intensity 6-Gal 4-Gal 4-Glc t-Gal Inositol t-Man 5-Ara(f) t-Glc 6-Glc 4-Xyl(p) t-Ara(p) 10 15 20 25 30 Time (minutes)

  3. C H D OA c OM e 118 205 M e O 162 161 OM e OA c 45 C H OM e 2 Primary Fragments Secondary Fragments + + C H O M e C H O M e H C O M e - HOAc H C OM e H C O A c H C C H O M e C H OM e 2 2 m/z 205 m/z 145 Primary and Secondary Fragments of the PMAA of Terminal Glucose

  4. Glycosyl Linkage Analysis 3,6-Gal t-Ara(f) 3-Gal Intensity 6-Gal 4-Gal 4-Glc t-Gal Inositol t-Man 5-Ara(f) t-Glc 6-Glc 4-Xyl(p) t-Ara(p) 10 15 20 25 30 Time (minutes)

  5. Glycosyl linkage analysis performed with an additional pre-reduction step 4-Glc Pre-6-glc t-Glc t-Rha 6-Glc Inositol 10 15 20 25 30 Time Minutes Peak Area

  6. Site Mapping on Glycoproteins • N-linked • O-linked

  7. N-linked Mapping

  8. 627.0 (M+2)/2 625.5 (M+H) 1253.1 1250.0 N-linked site mapping on an ion-trap with a mixture Of 016 and 018 Water

  9. (M+2)/2 of Peptide + 5 hexoses +1 +0 +2 +3 +4 * * * * * BEMAD 40ATTCTFSGSEASK54 # # Direct Fig. X: Site-Mapping of O-hexose sites on Peptides of PGC. Shown is the MS/MS spectra of a tryptic peptide from PGC. MS3 sequencing of 702.2 identified the peptide ATTCTFSGSEASK. Direct sequencing of PGC peptides identified two of the sites (#) while BEMAD site mapping identified all 5 sites of modification. In full, a total of 7 sites of O-Mannose modification (T42,T43, T45, S47, S49, S110, and S154) were identified for PGC.

  10. Figure 4: BEMAD Methodology. (A) Schematic of the b-elimination, Michael (conjugate) addition with DTT approach for replacing O-GlcNAc with a stable tag. (B) M+H of O-GlcNAc modified peptide before BEMAD and (C) M+H of peptide following chemistry by MALDI-TOF analysis. (D) DTT-modified peptide can be purified from a mixture using thiol-chromatography.

  11. Figure 6. ECD Fragmentation of O-GlcNAc Modified Peptide. Fragmentation of doubly charged parent ion of O-GlcNAc modified peptide on a LTQ-FT (Finnigan) using ECD fragmentation. Note mass accuracy of less than 2 ppm and Z7-Z12 and C7 ions still contain O-GlcNAc modified serine.

More Related