( ge pump Sample conditions: 50 ...)Characterization of Protein Oligosaccharides Using MSn

ge pump
Sample conditions: 50 ng/L total mixture in
50:50 MeOH/water, 1 mM sodium acetate
Capillary temperature: 200C
Needle voltage: +4.5-5 kV
Sheath gas: nitrogen, 30 unit
Collision energies: 60-65 units, except where noted


Figure 2. MS² of m/z 1169.5, the doubly-sodiated molecular ion of N₅H₅-I and N₅H₅-II.

Results

Figure 1a shows two oligosaccharides from the ovalbumin carbohydrate mixture. Since galactose and mannose are both hexoses, and therefore diastereomers of one another, both permethylated oligosaccharides have the same composition (HexNAc₅Hex₅) and molecular weight (2293 Da). With these two oligosaccharides infused together, MS² of their doubly-sodiated molecular ions at m/z 1169.5 (Figure 2) produces fragments corresponding to both N₅H₅-I and N₅H₅-II, with each oligosaccharide effectively contaminating the spectrum of the other.

The fragment at m/z 937.7 corresponds to loss of a Hex-HexNAc disaccharide antenna, which is a structural feature only found on N₅H₅-I. Therefore, selecting the m/z 937.7 ion and subjecting it to MS³ (Figure 3a) yields structural information on N5₅H₅-I, even as N₅H5₅-II is continuously infused into the instrument. Favorable cleavage sites lie adjacent to GlcNAc residues, particularly terminal GlcNAcs, resulting in the preferred fragmentation of glycosidic bonds (noted by B and Y labels) and suppression of cross-ring fragments (A and X-type ions).⁴ By performing MS<sup>n'"/>

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