Fig. 2: precisION localizes N-glycosylation on the dimeric human ACE2.
From: Uncovering hidden protein modifications with native top-down mass spectrometry
a, AlphaFold 3 structure of the ACE2 N432KO ectodomain dimer produced in HEK293 GNTI−/− cells. Man5GlcNAc2 glycans are displayed in blue. b, Mass spectrum of ACE2 N432KO (300 mM ammonium acetate, pH 7.0). An ensemble of ions assigned to the ACE2 dimer with 10–11 N-glycans and other unknown modifications (27+) were selected using the quadrupole and activated using ion–neutral collisions (HCD 130–200 V). The annotated MS2 spectrum generated at an HCD acceleration voltage of 150 V is displayed below. Inset are isotopic envelopes corresponding to sequence ions arising from distinctly modified forms of ACE2. c, Multinotch fragment-level open search used to identify the C terminus of the ACE2 construct. Variable proteolytic processing was observed. MS1- and MS2-based quantifications of the different truncated forms are shown inset. d, Fragment map illustrating the position of the fragmentation sites along the backbone of ACE2. Individual horizontal lines correspond to sequence ions assigned from the combined HCD dataset. N-glycosylation sites detected using bottom-up proteomics are displayed above the map. In some cases, we observed unmodified fragments enclosing glycosylation sites—this is likely due to the complete loss of the glycan upon activation. The right panel displays the positions of glycosylated and nonglycosylated sequence ions along the N terminus of ACE2. N-glycan sequons (N-X-S/T) are highlighted with dashed lines. e, Fragment-level open search results for the ACE2 dimer at different HCD acceleration voltages. Multiple significant offsets were observed. NGS, Asn-Gly-Ser; NIT, Asn-Ile-Thr; NLT, Asn-Leu-Thr.
