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.