Glycopeptide inclusion improves sequence coverage.
Addition of EThcD to facilitate the detection and identification of
glycopeptides during HDX-MS significantly improved sequence coverage and
conformational dynamic analysis of SARS-CoV-2 614G spike protein (Figure
S2). Glycopeptides were confidently identified at 9 out of the 22
potential occupied N-glycosylation sequons per monomer in spike 614G
variant (Table S1, 41% sequon coverage and 84% overall coverage) after
manual data processing. At 8 N-glycosylation sequons with confident
peptide coverage only glycopeptides were identified, with the exception,
N1074, covered by one non-glycosylated peptide (Figure 1C). Therefore,
without the inclusion of EThcD for glycopeptide detection and
identification, the sequon coverage would have dropped to 1 (5%) and
overall spike coverage to 76% (Figures 1 & S2). Additionally, because
nine N1074 glycopeptides had HexNAc(2)Hex(5) and HexNAc(2)Hex(6) glycans
(Figure 1), the detection of only the non-glycosylated peptide may not
represent the true conformational dynamics at N1074.
On-line digestion with nepenthesin-2 and pepsin allows analysis of
deuterated peptides in acidified conditions, but the non-specific nature
of digestion, along with the need for rapid digestion and LC separation,
brings concerns of lowered signal per peptide, especially when layered
with potential glycan structural diversity at each sequon. Despite these
potential issues with sensitivity, N-glycosylation sequon-specific
combinations of peptide and glycan diversity were observed (Figure 1).
Seven glycopeptides (one in 2+ and
3+ charge states) with unique amino acid sequences
including N234 displayed only one glycan type HexNAc(2)Hex(9). In
contrast, 13 unique glycopeptide amino acid sequences including N603
displayed five or more glycan types, while the only glycopeptide amino
acid sequence including N1134 displayed ten glycan types. Table S1
summarizes N-glycosylation sequon coverage and occupancy. All glycan
groups at the 9 sequons with coverage were of similar types
(oligomannose, complex, and hybrid) as described in previous
publications on spike microheterogeneity (34, 39, 46).
Evidence for deuterated glycopeptides is shown in Figures 2 to 4 using
glycopeptide 597-607 HexNAc(2)Hex(5) as an example. We obtained
high-confidence (Byonic score = 508.5) MS/MS identification (Figure 2),
reproducible extracted ion chromatograms (Figure 3A), and high-quality
isotopic envelopes (Figure 3B). We observed that each unique amino acid
sequence for a glycopeptide showed consistent uptake plots in both
control and heat denatured states although they had different N-glycan
groups. For example, glycopeptides with sequence597VITPGTN TSNQ607 had highly
consistent deuterium uptake (Figure 4, left peptide) even though 5
different glycan groups were identified on N603. However, overlay of
composite uptake plots for all twelve N603 overlapping glycopeptides
(Figure 4) indicated that unique amino acid sequences had different
uptakes, and increasing glycopeptide length affected estimated
deuteration. For example, C-terminally extending the above 597-607
glycopeptide by 2 amino acids decreased uptake in the control state but
increased uptake after heat-treatment (Figure 4, center peptide). Our
interpretation was that glycopeptide amino acid sequence at sequon N603
was a stronger determinant of deuteration than N-glycan identity.
Supporting this interpretation, glycopeptides covering sequons N61 and
N234 showed two levels of deuteration in the heat denatured state on
composite uptake plots, also associated with the length of
glycopeptides. For example, the N61 sequon’s proximity to the sequence66HAIH69 indicated that inclusion of
this histidine-rich “cool” spot of deuteration decreased the
percentage labeling of 7 longer glycopeptides ~15%
after heat treatment compared to 4 overlapping but shorter glycopeptides
that did not include the 66HAIH69sequence (Figure S3). This result is consistent with the slow
deuteration of His residues (47, 48), and amino acid sequence being a
stronger determinant of deuteration than N-glycan identity.