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. 2012 Apr:Chapter 12:12.11.1-12.11.11.
doi: 10.1002/0471140864.ps1211s68.

Use of CID/ETD mass spectrometry to analyze glycopeptides

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Use of CID/ETD mass spectrometry to analyze glycopeptides

Yehia Mechref. Curr Protoc Protein Sci. 2012 Apr.

Abstract

Collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) does not allow the characterization of glycopeptides because of the fragmentation of glycan structures and limited fragmentation of peptide backbones. Electron transfer dissociation (ETD) MS/MS, on the other hand, offers a complementary approach, prompting only peptide backbone fragmentation while keeping post-translational modifications intact. Characterization of glycopeptides using both CID and ETD is summarized in this unit. While CID provides information related to the composition of glycan moieties attached to a peptide backbone, ETD permits de novo sequencing of peptides. Radical anion transfer of electrons to the peptide backbone in ETD induces cleavage of the N-Cα bond. The glycan moiety is retained on the peptide backbone, largely unaffected by the ETD process, thus allowing the identification of the amino acid sequence of a glycopeptide and its glycosylation site. This unit discusses the use of both CID and ETD for better characterization of glycopeptides.

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Figures

Figure 1
Figure 1
CID (a) and ETD (b) tandem mass spectra of the doubly-glycosylated glycopeptide LYNFSNTGLPDPTLNTTYLQTLR derived from tryptically-digested horseradish peroxidase. The asterisk represents the peptide backbone. Symbols: blue square: GlcNAc, green circle: mannose, red triangle: fucose, orange star: xylose. Ions above 1000 m/z are doubly charged while those less than 1000 m/z are singly charged. All m/z values are presented as average mass. Both spectrum were acquired using HCT ULTRA with ETD (Brucker-Daltonics). The mass accuracy of this instrument in tandem MS is 0.8 Da. Reproduced with permission from (Alley, Mechref et al, 2009).
Figure 2
Figure 2
Glycopeptide analysis flow chart using a mass spectrometer capable of both acquiring CID and ETD spectra.
Figure 3
Figure 3
CID (a) and ETD (b) tandem MS of the tryptic glycopeptide RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR derived from bovine-fetuin with a triantennary trisialyated complex glycan. Ions above 1000 m/z are doubly charged while those less than 1000 m/z are singly charged. All m/z values are presented as average mass. Both spectrum were acquired using HCT ULTRA with ETD (Brucker-Daltonics). The mass accuracy of this instrument in tandem MS is 0.8 Da. Reproduced with permission from (Alley, Mechref et al, 2009).

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