Extended Data Fig. 7: N-glycans can be confidently identified and localized on intact, denatured proteins using top- down mass spectrometry with collisional activation.

a, Mass spectrum of Gallus gallus avidin acquired under denaturing and reducing conditions (50% MeCN, 1% formic acid in H₂O (v/v), 5 mM TCEP). An envelope of highly-charged proteoforms was selected (19+; m/z 830 ± 30) with the quadrupole and activated using ion–neutral collisions (HCD 10-15 V) to yield abundant sequence ions. The resulting MS² spectrum generated at an HCD acceleration voltage of 12.5 V is displayed below. Inset are isotopic envelopes corresponding to sequence ions that have retained a HexNAc moiety throughout fragmentation. b, Deconvoluted mass spectrum illustrating the distribution of avidin proteoforms existing in the sample. All assignments were made on the basis of findings from top-down MS measurements. c, Multinotch fragment-level open search used to identify avidin termini. Signal peptide cleavage can be observed along with variable C-terminal truncation. d, Fragment-level open search used to identify N-terminal modifications on avidin. b-type ions retaining HexNAc or large glycans can be observed. These ions were used to inform compositional assignments at the MS¹ level. e, Fragment map illustrating the position of the fragmentation sites along the backbone of avidin. Individual horizontal lines correspond to sequence ions assigned from the combined HCD dataset. Sequence ions retaining glycan structures are displayed in red. N-glycan sequons (N-X-S/T) are highlighted with dashed lines.