Isotopologue ratios of deuterated glycopeptide fragment ions.
Glycopeptide deuteration (Figures 2 to 4) and the presence of an amide bond in N-acetyl-hexosamines suggested that total glycopeptide deuteration included the N-acetyl amide group(s) of N-linked glycans, which has been previously confirmed at the glycan and glycopeptide levels (49) and recently reviewed (50). To measure N-acetyl hexose labeling, isotopologue ratios of glycopeptide597VITPGTN TSNQ607HexNAc(2)Hex(5) fragment ions were compared for both control and heat denatured states at all deuteration times. All MS/MS scans consistent with this glycopeptide precursor’s m/z and LC retention time were converted to lists of product ion m/z vs. intensity, and a ratio was taken between a fragment’s monoisotopic m/z (M0) and its M + 1 isotope’s m/z (M1, Figure 5 inset )). The bottom-up peptide detection strategy of the Tribrid Orbitrap Eclipse (see Experimental Section) limited the number of MS/MS scans of each deuterated glycopeptide to 3 to 6 per data file.
First, the theoretical M0/M1 ratio for the HexNAc product ion ([C8H14NO5]+, M0 204.0867 m/z ) is 11.5, similar to the observed 9.6 ± 2.9 ratio (Figure 5, C0 and H0, white bars). Second, the theoretical M0/M1 ratio for the ~y8 product ion ([C31H52N11O15]+, fragment 600PGTN TSNQ607without the glycan attached, Figure 1) is 2.99, similar to the observed 2.24 ± 1.21 ratio (Figure 5, C0 and H0, grey bars). This establishes that the product ion isotopologue analysis is appropriate, despite a limited number of MS/MS scans as described above. Third, the ~y8 product ion’s M0/M1 ratio significantly decreased after D2O exposure (Figure 5, grey bars), consistent with deuteration of this peptide’s 7 amide groups, reducing the amount of M0 818.3639 m/z and increasing the amount of M1 819.3779m/z . Fourth, the HexNAc product ion’s M0/M1 ratio also decreased after D2O exposure (Figure 5, white bars), indicating that it was labeled with deuterium. Student’s T-test of the HexNAc product ion’s M0/M1 ratios at 0 and 960 seconds of D2O exposure indicated a significant difference (p = 0.002), and these results collectively support deuteration at the amide proton in the N-acetyl moieties on glycans N-linked to glycopeptides. Corroborative data including two y-series product ions and HexNAc product ion for a different glycopeptide, 1132-1145 HexNAc(4)Hex(3)Fuc(1) is shown in Figure S4. An alternative explanation of this isotopologue analysis is the previously reported scrambling of deuterons between product ions during HCD (51, 52), and a different MS/MS strategy ((53, 54), beyond the scope of this study) would be needed to address that alternative, possibly including deconvolution of isotopic envelopes for product ions with 0, 1 and 2 deuterons.