Analysis of Site-Specific N-linked Glycopeptides for SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
Four 3.5-μg aliquots of SARS-CoV-2 S protein were reduced by incubating with 10 mM of dithiothreitol at 56°C and alkylated by 27.5 mM of iodoacetamide at room temperature in dark. The four aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, a combination of trypsin and Glu-C, or a combination of Glu-C and AspN. Three 10-μg aliquots of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark. The three aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, or a combination of trypsin and Lys-C. The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min. The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C. Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher-energy collisional dissociation (HCD) with stepped collision energy (15%, 25%, 35%) were collected in the Orbitrap at 15k resolution. pGlyco v2.2.2 (Liu et al., 2017) was used for database searches with mass tolerance set as 20 ppm for both precursors and fragments. The database search output was filtered to reach a 1% false discovery rate for glycans and 10% for peptides. Quantitation was performed by calculating spectral counts for each glycan composition at each site. Any N-linked glycan compositions identified by only one spectra were removed from quantitation. N-linked glycan compositions were categorized into 22 classes (including Unoccupied): HexNAc(2)Hex(9∼5)Fuc(0∼1) was classified as M9 to M5 respectively; HexNAc(2)Hex(4∼1)Fuc(0∼1) was classified as M1-M4; HexNAc(3∼6)Hex(5∼9)Fuc(0)NeuAc(0∼1) was classified as Hybrid with HexNAc(3∼6)Hex(5∼9)Fuc(1∼2)NeuAc(0∼1) classified as F-Hybrid; Complex-type glycans are classified based on the number of antenna, fucosylation, and sulfation: HexNAc(3)Hex(3∼4)Fuc(0)NeuAc(0∼1) is assigned as A1 with HexNAc(3)Hex(3∼4)Fuc(1∼2)NeuAc(0∼1) assigned as F-A1; HexNAc(4)Hex(3∼5)Fuc(0)NeuAc(0∼2) is assigned as A2/A1B with HexNAc(4)Hex(3∼5)Fuc(1∼5)NeuAc(0∼2) assigned as F-A2/A1B; HexNAc(5)Hex(3∼6)Fuc(0)NeuAc(0∼3) is assigned as A3/A2B with HexNAc(5)Hex(3∼6)Fuc(1∼3)NeuAc(0∼3) assigned as F-A3/A2B; HexNAc(6)Hex(3∼7)Fuc(0)NeuAc(0∼4) is assigned as A4/A3B with HexNAc(6)Hex(3∼7)Fuc(1∼3)NeuAc(0∼4) assigned as F-A4/A3B; HexNAc(7)Hex(3∼8)Fuc(0)NeuAc(0∼1) is assigned as A5/A4B with HexNAc(7)Hex(3∼8)Fuc(1∼3)NeuAc(0∼1) as F-A5/A4B; HexNAc(8)Hex(3∼9)Fuc(0) is assigned as A6/A5B with HexNAc(8)Hex(3∼9)Fuc(1) assigned as F-A6/A5B; any glycans identified with a sulfate are assigned as Sulfated.