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3 Results (m/z = 2312.2) and the partial peptide fragmentation. The peptide LYFSNVMLQDMQTDYSCNAR has three potential O-glycosylation sites and was found to be modified with two O-glycans: an O-mannose glycan (NeuAcHex 2 HexNAc) and a mucin-type glycan (NeuAcHexHexNAc). These were assigned based on the oxonium ions of the free glycans (for example the complete O-mannose glycan at m/z of 819) and the glycan fragmentation ions of the glycopeptides. However, the finding of the above mentioned glycopeptide is the only result obtained using the CID fragmentation approach. Therefore, protein fraction F15 from experiment (B) which showed a high amount of neurofascin based on the Western blot analysis was subjected to a glycopeptide analysis using the Orbitrap system. This LC-ESI system allows further fragmentation of the highest MS/MS signals thereby generating spectra with glycan fragmentation as well as peptide fragmentation (MS3). The proteins of F15 were digested with trypsin and GluC and the resulting peptides and glycopeptides were analyzed by LC-ESI-MS/MS. The Mascot search of these peptides (see Table 2) revealed that neurofascin was the predominant component. NCAM1 which also was present in the sample had already been shown not to carry the O-mannosyl modification. In addition, a few other proteins such as neurexin and tenascin-R were present which had not yet been analyzed for potential O-mannosylation. Therefore, glycopeptide spectra were discarded if the peptide mass could also correspond to one of these proteins in case no conclusive MS3 data were available. 39
3 Results Table 2: Protein identification based on Mascot results of protein fraction F15 (B). Protein (mouse) Score Neurofascin 443 Neural cell adhesion molecule 1 373 Neurexin-1-α 325 Sodium/potassium-transporting ATPase subunit α-1 297 Sodium/potassium-transporting ATPase subunit α-3 254 Tenascin-R 206 Neurexin-3-α 164 Neurocan core protein 161 Neural cell adhesion molecule L1-like protein 140 Sei<strong>zu</strong>re protein 6 104 Many identified peptides were highly modified, almost all were carbamylated and several dehydrated possibly due to the multistep purification procedure under harsh conditions. Several peptides modified with O-mannosyl glycans were found by searching for MS/MS spectra containing an m/z signal of 528. The peptide NNSPITD (amino acids 654−660 of NF, m/z = 760.4, modified by carbamylation and dehydration) was found to be O-mannosylated with an appropriate MS3 spectrum showing the unequivocal amino acid assignment (see Figure 16). The peptide was modified with Hex 4 HexNAc 4 Fuc and despite the presence of a consensus sequence not N-glycosylated since no typical fragmentation of core HexNAc could be observed. The fragmentation pattern instead revealed a modification with a mucin-type glycan (FucHex 2 HexNAc 3 ) at the serine residue and an O-mannosyl glycan (Hex 2 HexNAc) at the threonine residue as retrieved from the MS3 spectrum. 40
Page 1 and 2: Identification and Characterization
Page 3 and 4: I Index I Index of Contents I INDEX
Page 5 and 6: I Index FIGURE 13: WESTERN BLOT OF
Page 7 and 8: I Index I.III List of Abbreviations
Page 9 and 10: I Index PBS PMT PNN Phosphate Buffe
Page 11 and 12: I Index I.III.II Amino acids - thre
Page 13 and 14: I Index II Zusammenfassung Die O-Ma
Page 15 and 16: 1 Introduction glycosidically linke
Page 17 and 18: 1 Introduction glycans, complex-typ
Page 19 and 20: 1 Introduction O-glycosylation (typ
Page 21 and 22: 1 Introduction Figure 3: Schematic
Page 23 and 24: 1 Introduction the sites and functi
Page 25 and 26: 1 Introduction posttranslational pr
Page 27 and 28: 1 Introduction transfer of mannose
Page 29 and 30: 1 Introduction Together the above m
Page 31 and 32: 1 Introduction 1.4 Aim of this proj
Page 33 and 34: 2 Materials and methods Lysis and W
Page 35 and 36: 2 Materials and methods 2.1.3.2 Sec
Page 37 and 38: 2 Materials and methods 2.2.1.7 Fra
Page 39 and 40: 2 Materials and methods (see 2.2.2.
Page 41 and 42: Figure 9: O-Glycome of mouse brain.
Page 43 and 44: 3 Results Table 1: Sequential extra
Page 45 and 46: 3 Results that this protein might b
Page 47 and 48: 3 Results 3.2 O-Mannosylation on ne
Page 49 and 50: 3 Results identified by several 361
Page 51: 3 Results Figure 15: Fragmentation
Page 55 and 56: 3 Results Three other O-mannosyl gl
Page 57 and 58: 3 Results 3.3 O-Mannosylation of le
Page 59 and 60: 3 Results 3.3.2 Recombinant express
Page 61 and 62: 3 Results identified just as the gl
Page 63 and 64: 3 Results Subsequentially, the resu
Page 65 and 66: 3 Results In order to get more info
Page 67 and 68: 3 Results brain-specific protein ne
Page 69 and 70: 4 Summary and outlook malformations
Page 71 and 72: 4 Summary and outlook O-mannosylati
Page 73 and 74: 4 Summary and outlook Figure 24: Sc
Page 75 and 76: 5 References Bleckmann, C., Geyer,
Page 77 and 78: 5 References Frischknecht, R. & Sei
Page 79 and 80: 5 References Kriebel, M., Metzger,
Page 81 and 82: 5 References Nizet, V. & Esko, J. (
Page 83 and 84: 5 References Susuki, K. & Rasband,
Page 85 and 86: 6 Appendix 6 Appendix Table 6: Summ
Page 87 and 88: 6 Appendix Figure 27: Comparison be
Page 89 and 90: 6 Appendix Table 8: Protein identif
Page 91 and 92: 6 Appendix Figure 32: O-Glycan anal
Page 93 and 94: 6 Appendix Figure 34: ESI-MS/MS of
Page 95 and 96: 6 Appendix Figure 38: Analysis of t
Page 97 and 98: 6 Appendix Figure 40: Calf brain-de
Page 99 and 100: 6 Appendix Table 13: Protein identi
Page 101 and 102: 6 Appendix Table 14: Summary of hum
Page 103:
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