FIGURE LEGENDS
Figure 1 . Representative N-glycan microheterogeneity during
HDX-MS at spike sequons N61, N122, N801, N1074, and N1134. The
N-terminus (A), central region (B) and C-terminus (C) of spike are shown
including HDX peptide coverage (black horizontal lines), N-glycosylation
sequons (green horizontal dash within HDX peptide), signal peptide (grey
horizontal box), S2’ cleavage site (vertical red line), fusion peptide
(white horizontal box), and the locations of two disulfide bonds in
intact spike (orange brackets marked SS). Specific glycan structures
detected at each sequon are cartooned as HexNAc (blue squares), Hexose
(green or yellow circles), Fucose (red triangles) and NeuAc (purple
diamonds). Glycan structures are illustrative only and were not
determined in this study.
Figure 2. Tandem mass spectrum of glycopeptide 597-607
HexNAc(2)Hex(5) including sequon N603. Sample was unlabeled (0 sec
D2O exposure), fragment mass errors are shown in the
lower panel. Glycan product ions (green font) from HCD will trigger
EThcD MS/MS of a fresh precursor ion packet.
Figure 3. Extracted ion chromatograms (A) and isotopic
envelopes (B) of glycopeptide 597-607 HexNAc(2)Hex(5) including sequon
N603 (red N). Technical triplicate injections at each
D2O exposure time (central numbers) and both control
(black) and heat treatment (blue) conditions were injected in randomized
order.
Figure 4. Box and whiskers uptake plots of 12 glycopeptides
including sequon N603. The left, center, and right boxes are N = 6, 2,
and 4 unique glycopeptides (sequences, glycans, or charge states),
respectively. Dashed lines linking boxes indicate the same
D2O exposure time and control or heat denatured state, ×
indicates mean.
Figure 5. Isotopologue ratios of glycopeptide 597-607
HexNAc(2)Hex(5) fragment ions (see Figure 1 for a typical MS/MS spectrum
of this peptide). Inset : the ratio of M0 (monoisotopic peak) to
M1 (first M + 1 isotopic peak) was measured for HexNAc (204/205) and
~y8 (818/819) in a representative technical replicate at
each state and D2O exposure time. As product ions
shifted to a deuterated distribution the ratio decreased. N = 3 to 6
averaged MS/MS scans for each bar, error bars are one standard
deviation. At H240 and H960 the ~y8 M0 ion was not
detected.
Figure 6: Heat map data (960 sec) overlaid on a model based
upon PDBs 6VSB and 6VXX (55), view of central trimer head with stalk at
lower left corner. Without heat treatment (A) spike labeled between
0-37% indicated by white to red color. Regions with no coverage are
green. Taking the difference between heat treatment and control (B)
shows both decreased labeling (magenta color, one example is furin site)
and increased labeling (yellow color, trimer core is an example).
Regions with no coverage are blue.
Figure S1 . The effect of temperature and limited trypsin
digestion on the conformation of
SARS-CoV-2 spike protein (Omicron variant, Hexapro, His tag). Lane 1:
PageRuler Unstained
Protein Ladder, component MWs as indicated; Lane 2: empty; Lane 3: 0.5
μg spike, 25°C 1 hr,
trypsin digestion as described in Experimental; Lane 4: 0.5 μg spike,
35°C 1 hr, trypsin digestion as
described in Experimental; Lane 5: 0.5 μg spike, 45°C 1 hr, trypsin
digestion as described in Experimental; Lane 6: 0.5 μg spike, 55°C 1 hr,
trypsin digestion as described in Experimental; Lane 7: 0.5 μg spike,
65°C 1 hr, trypsin digestion as described in Experimental. Gel is a
non-reducing 4-12% Bis-Tris run in MES buffer at 100 V for 36 min,
bands were visualized with SYPRO Ruby Protein Gel Stain (Life
Technologies #S12000).
Figure S2 . Coverage map of 614G HexaPro Spike protein showing
561 peptides including 106 glycopeptides. Green font is protein coverage
(84%), green lines under N residues indicate glycosylation detected at
that N-glycosylation sequon (9 of 22).
Figure S3 . Distinct glycopeptide deuterium uptake patterns at
sequon N61 of SARS-CoV-2 spike protein. Data for all glycopeptides
covering N61 (top panel) shows higher uptake for the seven glycopeptides
not including sequence HAIH (lower right panel) compared to the four
glycopeptides including this sequence (lower left panel) after heat
treatment.
Figure S4 . Isotopologue ratios of glycopeptide1132IVNNTVYDPLQPEL1145HexNAc(4)Hex(3)Fuc(1) product ions. The ratio of isotopologue M0 to
isotopologue M1 was measured for HexNAc (204/205), ~y3
(358/359) and ~y6 (696/697) in 2 or 3 technical
replicates at each state and exposure time. As fragment ions shift to a
deuterated distribution the ratio decreases. N = 3 to 6 averaged MS/MS
scans for each bar.