Id |
Subject |
Object |
Predicate |
Lexical cue |
T174 |
0-34 |
Sentence |
denotes |
Mass spectrometry of glycopeptides |
T175 |
35-142 |
Sentence |
denotes |
Aliquots of 30–50 μg of coronavirus spikes were denatured, reduced and alkylated as described previously36. |
T176 |
143-287 |
Sentence |
denotes |
Proteins were proteolytically digested with trypsin (Promega), chymotrypsin (Promega), alpha-lytic protease (Sigma-Aldrich) and Glu-C (Promega). |
T177 |
288-431 |
Sentence |
denotes |
Reaction mixtures were dried and peptides/glycopeptides were extracted using C18 Zip-tip (MerckMilipore) following the manufacturer’s protocol. |
T178 |
432-643 |
Sentence |
denotes |
Samples were resuspended in 0.1% formic acid prior to analysis by liquid chromatography-mass spectrometry using an Easy-nLC 1200 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). |
T179 |
644-872 |
Sentence |
denotes |
Glycopeptides were separated using an EasySpray PepMap RSLC C18 column (75 μm × 75 cm) with a 240-min linear solvent gradient of 0–32% acetonitrile in 0.1% formic acid, followed by 35 min of 80% acetonitrile in 0.1% formic acid. |
T180 |
873-1020 |
Sentence |
denotes |
Other settings include an LC flow rate of 200 nL/min, spray voltage of 2.8 kV, capillary temperature of 275 °C, and an HCD collision energy of 50%. |
T181 |
1021-1152 |
Sentence |
denotes |
Precursor and fragmentation detection were performed using an Orbitrap at the following resolution: MS1 = 100,000 and MS2 = 30,000. |
T182 |
1153-1271 |
Sentence |
denotes |
The automatic gain control (AGC) targets were MS1 = 4e5 and MS2 = 5e4, and injection times were MS1 = 50 and MS2 = 54. |
T183 |
1272-1414 |
Sentence |
denotes |
The following cleavage sites were used for the respective proteases; trypsin=R/K, chymotrypsin=F/Y/W, alpha lytic protease=T/A/S/V, Glu C=E/D. |
T184 |
1415-1456 |
Sentence |
denotes |
Number of missed cleavages were set at 3. |
T185 |
1457-1504 |
Sentence |
denotes |
The following modifications were also included: |
T186 |
1505-1802 |
Sentence |
denotes |
Carbamidomethyl (+57.021464, target=C, fine control=fixed), Oxidation (+15.994915, target=M, fine control=variable rare 1), Glu to pyro-Glu (−18.010565, target=peptide N-term E, fine control=variable rare 1), and Gln to pyro-Glu (−17.026549, target peptide N-term Q, fine control=variable rare 1). |
T187 |
1803-1950 |
Sentence |
denotes |
Glycopeptide fragmentation data were extracted form raw files using ByonicTM (Version 3.5.0) and ByologicTM (Version 3.5-15; Protein Metrics Inc.). |
T188 |
1951-2162 |
Sentence |
denotes |
Glycopeptide fragmentation data were manually evaluated with true-positive assignments given when correct b- and y-fragments and oxonium ions corresponding to the peptide and glycan, respectively, were observed. |
T189 |
2163-2257 |
Sentence |
denotes |
The precursor mass tolerance was set at 4 ppm for precursor ions and 10 ppm for fragment ions. |
T190 |
2258-2351 |
Sentence |
denotes |
MS data were searched using a glycan library (SI Fig. 9) with the identical peptide sequence. |
T191 |
2352-2396 |
Sentence |
denotes |
A 1% false discovery rate (FDR) was applied. |
T192 |
2397-2614 |
Sentence |
denotes |
The extracted ion chromatographic areas for each true-positive glycopeptide, with the same amino-acid sequence, were compared to determine the relative quantitation of glycoforms at each specific N-linked glycan site. |